http://2009.igem.org/wiki/index.php?title=Special:Contributions/Eniak&feed=atom&limit=50&target=Eniak&year=&month=2009.igem.org - User contributions [en]2024-03-29T08:57:24ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/AugustTeam:IPN-UNAM-Mexico/Notebook/August2009-10-21T07:44:44Z<p>Eniak: /* 06-Agust-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''03-Agust-2009'''''===<br />
<br />
Results of previous transformations: <br />
<br />
<center><br />
{|<br />
|F1610<br />
|<br />
|Worked<br />
|-<br />
|K091146<br />
|<br />
|Worked<br />
|-<br />
|K081016<br />
|<br />
|Worked<br />
|-<br />
|K081009<br />
|<br />
|Didn't work<br />
|-<br />
|R0051<br />
|<br />
|Worked<br />
|-<br />
|K081018<br />
|<br />
|Didn't work<br />
|}<br />
</center><br />
----<br />
----<br />
<br />
==='''''04-Agust-2009'''''===<br />
<br />
Only the transformed cells from yesterday's experiments are kept in incubation, being prepared for tomorrow’s extraction. <br />
<br />
----<br />
----<br />
<br />
==='''''05-Agust-2009'''''===<br />
<br />
<br />
We made mini preps ( see protocol in protocols section) and did the follow restrictions:<br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
!Restriction<br />
|-<br />
|2<br />
|BBa_R0079<br />
|S & P<br />
|-<br />
|3<br />
|BBa_F1610<br />
|X & P<br />
|-<br />
|4<br />
|BBa_K091146<br />
|S & P<br />
|-<br />
|5<br />
|BBa_K093005<br />
|X & P<br />
|-<br />
|6<br />
|BBa_K081016<br />
|X & P<br />
|-<br />
|7<br />
|BBa_EC840<br />
|X & P<br />
|-<br />
|8<br />
|BBa_K081009<br />
|X & P<br />
|-<br />
|9<br />
|BBa_R0051<br />
|S &P<br />
|-<br />
|10<br />
|BBa_J06800<br />
|X & P<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|X & P<br />
|-<br />
|12<br />
|BBa_B0034<br />
|S & P<br />
|-<br />
|13<br />
|BBa_C0079<br />
|E & S<br />
|-<br />
|14<br />
|BBa_C0179<br />
|E & S<br />
|-<br />
|15<br />
|BBa_B0015<br />
|E & X<br />
|-<br />
|16<br />
|BBa_K081018<br />
|E & S<br />
|-<br />
|17<br />
|BBa_K116640<br />
|X & P<br />
|-<br />
|23<br />
|BBa_S03154<br />
|X & P<br />
|-<br />
|24<br />
|BBa_k145201<br />
|E & S<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''06-Agust-2009'''''===<br />
<br />
* We prepared 5ml falcon tubes containing liquid agar and Ampr for inoculation.<br />
*We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.<br />
<br />
----<br />
----<br />
<br />
==='''''07-Agust-2009'''''===<br />
<br />
*Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.<br />
<br />
----<br />
----<br />
<br />
==='''''10-Agust-2009'''''===<br />
<br />
We made DNA purification from the Gel an we are planing start ligations this week.<br />
<br />
----<br />
----<br />
<br />
==='''''11-Agust-2009'''''===<br />
<br />
*Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations. <br />
<br />
----<br />
----<br />
<br />
==='''''12-Agust-2009'''''===<br />
<br />
We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).<br />
<br />
In this step we use 2 as backbone and 3 as insert, on the same 15 as backbone and 13 as insert, for 2-3 (BBa_K266000) we expect 3034 bp band as shown below, for 15-13 4074 bp. 4 as backbone 23 as insert. For ligation we use purifed DNA from agarosa Gel.<br />
<br />
<br />
<center><br />
{| border="0.5"<br />
! Mix Ligation <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 5μl<br />
|-<br />
|Insert DNA<br />
|align="right" | 25μl<br />
|-<br />
|T4 DNA Ligasa Buffer<br />
|align="right" | 3μl<br />
|-<br />
|T4 DNA ligasa <br />
|align="right" | 2μl<br />
|-<br />
|ATP<br />
|align="right" | 3μl<br />
|}<br />
</center><br />
<br />
The ligation left 17 hours overnight in 14ºC.<br />
<br />
----<br />
----<br />
<br />
==='''''13-Agust-2009'''''===<br />
<br />
We transform the ligation by thermic shock and plate.<br />
We received primers for 11 and 12.<br />
<br />
----<br />
----<br />
<br />
==='''''14-Agust-2009'''''===<br />
<br />
The transformation was successful we get colonies, we picket out for plasmid and left in 37ºC overnight.<br />
<br />
----<br />
----<br />
<br />
==='''''15-Agust-2009'''''===<br />
<br />
Only take off culture from camera and prepare for monday mini prep.<br />
<br />
----<br />
----<br />
<br />
==='''''17-Agust-2009'''''===<br />
<br />
We did mini prep and run out on a gel, we expect 3034 bp for L2_3(BBa_K266000) and 4074 bp for L15_13 (Bba_K266002) this are successful not so for 4_23 this is unsuccessful and proceed to repeat the ligation.<br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''18-Agust-2009'''''===<br />
<br />
The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight) <br />
<br />
----<br />
----<br />
<br />
==='''''19-Agust-2009'''''===<br />
<br />
Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.<br />
<br />
----<br />
----<br />
<br />
==='''''20-Agust-20009'''''===<br />
<br />
Purificated from gel for begin ligation.<br />
<br />
----<br />
----<br />
<br />
==='''''21-Agust-2009'''''===<br />
<br />
The transformation was through thermick shok and plate the weight we expect is amplicon 1515 bp 15_13 4074 bp total for Bba_K266003 5574 bp. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''22-Agust-2009'''''===<br />
<br />
The transformation was successful and we get colonies picket out for midi prep.<br />
For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.<br />
<br />
----<br />
----<br />
<br />
==='''''23-Agust-2009'''''===<br />
<br />
Meeting; we are detected a mistake on the primer, our primer musted include J23100 promotor now we have to do a extra ligation, and use J23100 as backbone.<br />
<br />
----<br />
----<br />
<br />
==='''''24-Agust-2009'''''===<br />
<br />
We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight<br />
<br />
----<br />
----<br />
<br />
==='''''25-Agust-2009'''''===<br />
<br />
Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
For ligation we going to do a mix and leave 17 hours in 14ºC camera.<br />
<br />
----<br />
----<br />
<br />
==='''''26-Agust-2009'''''===<br />
<br />
We transform by thermic shock and plate.<br />
<br />
----<br />
----<br />
<br />
==='''''27-Agust-2009'''''===<br />
<br />
The transformation was unsuccessful we haven't colonies.<br />
We going to repeat the ligation and left overnight incubation 14ºC.<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/AugustTeam:IPN-UNAM-Mexico/Notebook/August2009-10-21T07:41:37Z<p>Eniak: /* 05-Agust-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''03-Agust-2009'''''===<br />
<br />
Results of previous transformations: <br />
<br />
<center><br />
{|<br />
|F1610<br />
|<br />
|Worked<br />
|-<br />
|K091146<br />
|<br />
|Worked<br />
|-<br />
|K081016<br />
|<br />
|Worked<br />
|-<br />
|K081009<br />
|<br />
|Didn't work<br />
|-<br />
|R0051<br />
|<br />
|Worked<br />
|-<br />
|K081018<br />
|<br />
|Didn't work<br />
|}<br />
</center><br />
----<br />
----<br />
<br />
==='''''04-Agust-2009'''''===<br />
<br />
Only the transformed cells from yesterday's experiments are kept in incubation, being prepared for tomorrow’s extraction. <br />
<br />
----<br />
----<br />
<br />
==='''''05-Agust-2009'''''===<br />
<br />
<br />
We made mini preps ( see protocol in protocols section) and did the follow restrictions:<br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
!Restriction<br />
|-<br />
|2<br />
|BBa_R0079<br />
|S & P<br />
|-<br />
|3<br />
|BBa_F1610<br />
|X & P<br />
|-<br />
|4<br />
|BBa_K091146<br />
|S & P<br />
|-<br />
|5<br />
|BBa_K093005<br />
|X & P<br />
|-<br />
|6<br />
|BBa_K081016<br />
|X & P<br />
|-<br />
|7<br />
|BBa_EC840<br />
|X & P<br />
|-<br />
|8<br />
|BBa_K081009<br />
|X & P<br />
|-<br />
|9<br />
|BBa_R0051<br />
|S &P<br />
|-<br />
|10<br />
|BBa_J06800<br />
|X & P<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|X & P<br />
|-<br />
|12<br />
|BBa_B0034<br />
|S & P<br />
|-<br />
|13<br />
|BBa_C0079<br />
|E & S<br />
|-<br />
|14<br />
|BBa_C0179<br />
|E & S<br />
|-<br />
|15<br />
|BBa_B0015<br />
|E & X<br />
|-<br />
|16<br />
|BBa_K081018<br />
|E & S<br />
|-<br />
|17<br />
|BBa_K116640<br />
|X & P<br />
|-<br />
|23<br />
|BBa_S03154<br />
|X & P<br />
|-<br />
|24<br />
|BBa_k145201<br />
|E & S<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''06-Agust-2009'''''===<br />
<br />
* Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.<br />
*We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.<br />
<br />
----<br />
----<br />
<br />
==='''''07-Agust-2009'''''===<br />
<br />
*Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.<br />
<br />
----<br />
----<br />
<br />
==='''''10-Agust-2009'''''===<br />
<br />
We made DNA purification from the Gel an we are planing start ligations this week.<br />
<br />
----<br />
----<br />
<br />
==='''''11-Agust-2009'''''===<br />
<br />
*Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations. <br />
<br />
----<br />
----<br />
<br />
==='''''12-Agust-2009'''''===<br />
<br />
We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).<br />
<br />
In this step we use 2 as backbone and 3 as insert, on the same 15 as backbone and 13 as insert, for 2-3 (BBa_K266000) we expect 3034 bp band as shown below, for 15-13 4074 bp. 4 as backbone 23 as insert. For ligation we use purifed DNA from agarosa Gel.<br />
<br />
<br />
<center><br />
{| border="0.5"<br />
! Mix Ligation <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 5μl<br />
|-<br />
|Insert DNA<br />
|align="right" | 25μl<br />
|-<br />
|T4 DNA Ligasa Buffer<br />
|align="right" | 3μl<br />
|-<br />
|T4 DNA ligasa <br />
|align="right" | 2μl<br />
|-<br />
|ATP<br />
|align="right" | 3μl<br />
|}<br />
</center><br />
<br />
The ligation left 17 hours overnight in 14ºC.<br />
<br />
----<br />
----<br />
<br />
==='''''13-Agust-2009'''''===<br />
<br />
We transform the ligation by thermic shock and plate.<br />
We received primers for 11 and 12.<br />
<br />
----<br />
----<br />
<br />
==='''''14-Agust-2009'''''===<br />
<br />
The transformation was successful we get colonies, we picket out for plasmid and left in 37ºC overnight.<br />
<br />
----<br />
----<br />
<br />
==='''''15-Agust-2009'''''===<br />
<br />
Only take off culture from camera and prepare for monday mini prep.<br />
<br />
----<br />
----<br />
<br />
==='''''17-Agust-2009'''''===<br />
<br />
We did mini prep and run out on a gel, we expect 3034 bp for L2_3(BBa_K266000) and 4074 bp for L15_13 (Bba_K266002) this are successful not so for 4_23 this is unsuccessful and proceed to repeat the ligation.<br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''18-Agust-2009'''''===<br />
<br />
The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight) <br />
<br />
----<br />
----<br />
<br />
==='''''19-Agust-2009'''''===<br />
<br />
Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.<br />
<br />
----<br />
----<br />
<br />
==='''''20-Agust-20009'''''===<br />
<br />
Purificated from gel for begin ligation.<br />
<br />
----<br />
----<br />
<br />
==='''''21-Agust-2009'''''===<br />
<br />
The transformation was through thermick shok and plate the weight we expect is amplicon 1515 bp 15_13 4074 bp total for Bba_K266003 5574 bp. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''22-Agust-2009'''''===<br />
<br />
The transformation was successful and we get colonies picket out for midi prep.<br />
For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.<br />
<br />
----<br />
----<br />
<br />
==='''''23-Agust-2009'''''===<br />
<br />
Meeting; we are detected a mistake on the primer, our primer musted include J23100 promotor now we have to do a extra ligation, and use J23100 as backbone.<br />
<br />
----<br />
----<br />
<br />
==='''''24-Agust-2009'''''===<br />
<br />
We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight<br />
<br />
----<br />
----<br />
<br />
==='''''25-Agust-2009'''''===<br />
<br />
Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
For ligation we going to do a mix and leave 17 hours in 14ºC camera.<br />
<br />
----<br />
----<br />
<br />
==='''''26-Agust-2009'''''===<br />
<br />
We transform by thermic shock and plate.<br />
<br />
----<br />
----<br />
<br />
==='''''27-Agust-2009'''''===<br />
<br />
The transformation was unsuccessful we haven't colonies.<br />
We going to repeat the ligation and left overnight incubation 14ºC.<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/AugustTeam:IPN-UNAM-Mexico/Notebook/August2009-10-21T07:40:44Z<p>Eniak: /* 04-Agust-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''03-Agust-2009'''''===<br />
<br />
Results of previous transformations: <br />
<br />
<center><br />
{|<br />
|F1610<br />
|<br />
|Worked<br />
|-<br />
|K091146<br />
|<br />
|Worked<br />
|-<br />
|K081016<br />
|<br />
|Worked<br />
|-<br />
|K081009<br />
|<br />
|Didn't work<br />
|-<br />
|R0051<br />
|<br />
|Worked<br />
|-<br />
|K081018<br />
|<br />
|Didn't work<br />
|}<br />
</center><br />
----<br />
----<br />
<br />
==='''''04-Agust-2009'''''===<br />
<br />
Only the transformed cells from yesterday's experiments are kept in incubation, being prepared for tomorrow’s extraction. <br />
<br />
----<br />
----<br />
<br />
==='''''05-Agust-2009'''''===<br />
<br />
<br />
We made mini preped (protocol) and did the follow restrictions:<br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
!Restriction<br />
|-<br />
|2<br />
|BBa_R0079<br />
|S & P<br />
|-<br />
|3<br />
|BBa_F1610<br />
|X & P<br />
|-<br />
|4<br />
|BBa_K091146<br />
|S & P<br />
|-<br />
|5<br />
|BBa_K093005<br />
|X & P<br />
|-<br />
|6<br />
|BBa_K081016<br />
|X & P<br />
|-<br />
|7<br />
|BBa_EC840<br />
|X & P<br />
|-<br />
|8<br />
|BBa_K081009<br />
|X & P<br />
|-<br />
|9<br />
|BBa_R0051<br />
|S &P<br />
|-<br />
|10<br />
|BBa_J06800<br />
|X & P<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|X & P<br />
|-<br />
|12<br />
|BBa_B0034<br />
|S & P<br />
|-<br />
|13<br />
|BBa_C0079<br />
|E & S<br />
|-<br />
|14<br />
|BBa_C0179<br />
|E & S<br />
|-<br />
|15<br />
|BBa_B0015<br />
|E & X<br />
|-<br />
|16<br />
|BBa_K081018<br />
|E & S<br />
|-<br />
|17<br />
|BBa_K116640<br />
|X & P<br />
|-<br />
|23<br />
|BBa_S03154<br />
|X & P<br />
|-<br />
|24<br />
|BBa_k145201<br />
|E & S<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''06-Agust-2009'''''===<br />
<br />
* Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.<br />
*We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.<br />
<br />
----<br />
----<br />
<br />
==='''''07-Agust-2009'''''===<br />
<br />
*Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.<br />
<br />
----<br />
----<br />
<br />
==='''''10-Agust-2009'''''===<br />
<br />
We made DNA purification from the Gel an we are planing start ligations this week.<br />
<br />
----<br />
----<br />
<br />
==='''''11-Agust-2009'''''===<br />
<br />
*Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations. <br />
<br />
----<br />
----<br />
<br />
==='''''12-Agust-2009'''''===<br />
<br />
We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).<br />
<br />
In this step we use 2 as backbone and 3 as insert, on the same 15 as backbone and 13 as insert, for 2-3 (BBa_K266000) we expect 3034 bp band as shown below, for 15-13 4074 bp. 4 as backbone 23 as insert. For ligation we use purifed DNA from agarosa Gel.<br />
<br />
<br />
<center><br />
{| border="0.5"<br />
! Mix Ligation <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 5μl<br />
|-<br />
|Insert DNA<br />
|align="right" | 25μl<br />
|-<br />
|T4 DNA Ligasa Buffer<br />
|align="right" | 3μl<br />
|-<br />
|T4 DNA ligasa <br />
|align="right" | 2μl<br />
|-<br />
|ATP<br />
|align="right" | 3μl<br />
|}<br />
</center><br />
<br />
The ligation left 17 hours overnight in 14ºC.<br />
<br />
----<br />
----<br />
<br />
==='''''13-Agust-2009'''''===<br />
<br />
We transform the ligation by thermic shock and plate.<br />
We received primers for 11 and 12.<br />
<br />
----<br />
----<br />
<br />
==='''''14-Agust-2009'''''===<br />
<br />
The transformation was successful we get colonies, we picket out for plasmid and left in 37ºC overnight.<br />
<br />
----<br />
----<br />
<br />
==='''''15-Agust-2009'''''===<br />
<br />
Only take off culture from camera and prepare for monday mini prep.<br />
<br />
----<br />
----<br />
<br />
==='''''17-Agust-2009'''''===<br />
<br />
We did mini prep and run out on a gel, we expect 3034 bp for L2_3(BBa_K266000) and 4074 bp for L15_13 (Bba_K266002) this are successful not so for 4_23 this is unsuccessful and proceed to repeat the ligation.<br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''18-Agust-2009'''''===<br />
<br />
The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight) <br />
<br />
----<br />
----<br />
<br />
==='''''19-Agust-2009'''''===<br />
<br />
Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.<br />
<br />
----<br />
----<br />
<br />
==='''''20-Agust-20009'''''===<br />
<br />
Purificated from gel for begin ligation.<br />
<br />
----<br />
----<br />
<br />
==='''''21-Agust-2009'''''===<br />
<br />
The transformation was through thermick shok and plate the weight we expect is amplicon 1515 bp 15_13 4074 bp total for Bba_K266003 5574 bp. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''22-Agust-2009'''''===<br />
<br />
The transformation was successful and we get colonies picket out for midi prep.<br />
For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.<br />
<br />
----<br />
----<br />
<br />
==='''''23-Agust-2009'''''===<br />
<br />
Meeting; we are detected a mistake on the primer, our primer musted include J23100 promotor now we have to do a extra ligation, and use J23100 as backbone.<br />
<br />
----<br />
----<br />
<br />
==='''''24-Agust-2009'''''===<br />
<br />
We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight<br />
<br />
----<br />
----<br />
<br />
==='''''25-Agust-2009'''''===<br />
<br />
Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
For ligation we going to do a mix and leave 17 hours in 14ºC camera.<br />
<br />
----<br />
----<br />
<br />
==='''''26-Agust-2009'''''===<br />
<br />
We transform by thermic shock and plate.<br />
<br />
----<br />
----<br />
<br />
==='''''27-Agust-2009'''''===<br />
<br />
The transformation was unsuccessful we haven't colonies.<br />
We going to repeat the ligation and left overnight incubation 14ºC.<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/AugustTeam:IPN-UNAM-Mexico/Notebook/August2009-10-21T07:40:20Z<p>Eniak: /* 04-Agust-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''03-Agust-2009'''''===<br />
<br />
Results of previous transformations: <br />
<br />
<center><br />
{|<br />
|F1610<br />
|<br />
|Worked<br />
|-<br />
|K091146<br />
|<br />
|Worked<br />
|-<br />
|K081016<br />
|<br />
|Worked<br />
|-<br />
|K081009<br />
|<br />
|Didn't work<br />
|-<br />
|R0051<br />
|<br />
|Worked<br />
|-<br />
|K081018<br />
|<br />
|Didn't work<br />
|}<br />
</center><br />
----<br />
----<br />
<br />
==='''''04-Agust-2009'''''===<br />
<br />
Yesterday’s Only the transformed cells from yesterday's experiments are kept in incubation, being prepared for tomorrow’s extraction. <br />
<br />
----<br />
----<br />
<br />
==='''''05-Agust-2009'''''===<br />
<br />
<br />
We made mini preped (protocol) and did the follow restrictions:<br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
!Restriction<br />
|-<br />
|2<br />
|BBa_R0079<br />
|S & P<br />
|-<br />
|3<br />
|BBa_F1610<br />
|X & P<br />
|-<br />
|4<br />
|BBa_K091146<br />
|S & P<br />
|-<br />
|5<br />
|BBa_K093005<br />
|X & P<br />
|-<br />
|6<br />
|BBa_K081016<br />
|X & P<br />
|-<br />
|7<br />
|BBa_EC840<br />
|X & P<br />
|-<br />
|8<br />
|BBa_K081009<br />
|X & P<br />
|-<br />
|9<br />
|BBa_R0051<br />
|S &P<br />
|-<br />
|10<br />
|BBa_J06800<br />
|X & P<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|X & P<br />
|-<br />
|12<br />
|BBa_B0034<br />
|S & P<br />
|-<br />
|13<br />
|BBa_C0079<br />
|E & S<br />
|-<br />
|14<br />
|BBa_C0179<br />
|E & S<br />
|-<br />
|15<br />
|BBa_B0015<br />
|E & X<br />
|-<br />
|16<br />
|BBa_K081018<br />
|E & S<br />
|-<br />
|17<br />
|BBa_K116640<br />
|X & P<br />
|-<br />
|23<br />
|BBa_S03154<br />
|X & P<br />
|-<br />
|24<br />
|BBa_k145201<br />
|E & S<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''06-Agust-2009'''''===<br />
<br />
* Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.<br />
*We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.<br />
<br />
----<br />
----<br />
<br />
==='''''07-Agust-2009'''''===<br />
<br />
*Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.<br />
<br />
----<br />
----<br />
<br />
==='''''10-Agust-2009'''''===<br />
<br />
We made DNA purification from the Gel an we are planing start ligations this week.<br />
<br />
----<br />
----<br />
<br />
==='''''11-Agust-2009'''''===<br />
<br />
*Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations. <br />
<br />
----<br />
----<br />
<br />
==='''''12-Agust-2009'''''===<br />
<br />
We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).<br />
<br />
In this step we use 2 as backbone and 3 as insert, on the same 15 as backbone and 13 as insert, for 2-3 (BBa_K266000) we expect 3034 bp band as shown below, for 15-13 4074 bp. 4 as backbone 23 as insert. For ligation we use purifed DNA from agarosa Gel.<br />
<br />
<br />
<center><br />
{| border="0.5"<br />
! Mix Ligation <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 5μl<br />
|-<br />
|Insert DNA<br />
|align="right" | 25μl<br />
|-<br />
|T4 DNA Ligasa Buffer<br />
|align="right" | 3μl<br />
|-<br />
|T4 DNA ligasa <br />
|align="right" | 2μl<br />
|-<br />
|ATP<br />
|align="right" | 3μl<br />
|}<br />
</center><br />
<br />
The ligation left 17 hours overnight in 14ºC.<br />
<br />
----<br />
----<br />
<br />
==='''''13-Agust-2009'''''===<br />
<br />
We transform the ligation by thermic shock and plate.<br />
We received primers for 11 and 12.<br />
<br />
----<br />
----<br />
<br />
==='''''14-Agust-2009'''''===<br />
<br />
The transformation was successful we get colonies, we picket out for plasmid and left in 37ºC overnight.<br />
<br />
----<br />
----<br />
<br />
==='''''15-Agust-2009'''''===<br />
<br />
Only take off culture from camera and prepare for monday mini prep.<br />
<br />
----<br />
----<br />
<br />
==='''''17-Agust-2009'''''===<br />
<br />
We did mini prep and run out on a gel, we expect 3034 bp for L2_3(BBa_K266000) and 4074 bp for L15_13 (Bba_K266002) this are successful not so for 4_23 this is unsuccessful and proceed to repeat the ligation.<br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''18-Agust-2009'''''===<br />
<br />
The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight) <br />
<br />
----<br />
----<br />
<br />
==='''''19-Agust-2009'''''===<br />
<br />
Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.<br />
<br />
----<br />
----<br />
<br />
==='''''20-Agust-20009'''''===<br />
<br />
Purificated from gel for begin ligation.<br />
<br />
----<br />
----<br />
<br />
==='''''21-Agust-2009'''''===<br />
<br />
The transformation was through thermick shok and plate the weight we expect is amplicon 1515 bp 15_13 4074 bp total for Bba_K266003 5574 bp. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''22-Agust-2009'''''===<br />
<br />
The transformation was successful and we get colonies picket out for midi prep.<br />
For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.<br />
<br />
----<br />
----<br />
<br />
==='''''23-Agust-2009'''''===<br />
<br />
Meeting; we are detected a mistake on the primer, our primer musted include J23100 promotor now we have to do a extra ligation, and use J23100 as backbone.<br />
<br />
----<br />
----<br />
<br />
==='''''24-Agust-2009'''''===<br />
<br />
We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight<br />
<br />
----<br />
----<br />
<br />
==='''''25-Agust-2009'''''===<br />
<br />
Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
For ligation we going to do a mix and leave 17 hours in 14ºC camera.<br />
<br />
----<br />
----<br />
<br />
==='''''26-Agust-2009'''''===<br />
<br />
We transform by thermic shock and plate.<br />
<br />
----<br />
----<br />
<br />
==='''''27-Agust-2009'''''===<br />
<br />
The transformation was unsuccessful we haven't colonies.<br />
We going to repeat the ligation and left overnight incubation 14ºC.<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/AugustTeam:IPN-UNAM-Mexico/Notebook/August2009-10-21T07:28:29Z<p>Eniak: /* 03-Agust-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''03-Agust-2009'''''===<br />
<br />
Results of previous transformations: <br />
<br />
<center><br />
{|<br />
|F1610<br />
|<br />
|Worked<br />
|-<br />
|K091146<br />
|<br />
|Worked<br />
|-<br />
|K081016<br />
|<br />
|Worked<br />
|-<br />
|K081009<br />
|<br />
|Didn't work<br />
|-<br />
|R0051<br />
|<br />
|Worked<br />
|-<br />
|K081018<br />
|<br />
|Didn't work<br />
|}<br />
</center><br />
----<br />
----<br />
<br />
==='''''04-Agust-2009'''''===<br />
<br />
Yesterday’s transformations were successful, continued to incubate it will ready for tomorrow’s mini prepped. <br />
<br />
----<br />
----<br />
<br />
==='''''05-Agust-2009'''''===<br />
<br />
<br />
We made mini preped (protocol) and did the follow restrictions:<br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
!Restriction<br />
|-<br />
|2<br />
|BBa_R0079<br />
|S & P<br />
|-<br />
|3<br />
|BBa_F1610<br />
|X & P<br />
|-<br />
|4<br />
|BBa_K091146<br />
|S & P<br />
|-<br />
|5<br />
|BBa_K093005<br />
|X & P<br />
|-<br />
|6<br />
|BBa_K081016<br />
|X & P<br />
|-<br />
|7<br />
|BBa_EC840<br />
|X & P<br />
|-<br />
|8<br />
|BBa_K081009<br />
|X & P<br />
|-<br />
|9<br />
|BBa_R0051<br />
|S &P<br />
|-<br />
|10<br />
|BBa_J06800<br />
|X & P<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|X & P<br />
|-<br />
|12<br />
|BBa_B0034<br />
|S & P<br />
|-<br />
|13<br />
|BBa_C0079<br />
|E & S<br />
|-<br />
|14<br />
|BBa_C0179<br />
|E & S<br />
|-<br />
|15<br />
|BBa_B0015<br />
|E & X<br />
|-<br />
|16<br />
|BBa_K081018<br />
|E & S<br />
|-<br />
|17<br />
|BBa_K116640<br />
|X & P<br />
|-<br />
|23<br />
|BBa_S03154<br />
|X & P<br />
|-<br />
|24<br />
|BBa_k145201<br />
|E & S<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''06-Agust-2009'''''===<br />
<br />
* Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.<br />
*We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.<br />
<br />
----<br />
----<br />
<br />
==='''''07-Agust-2009'''''===<br />
<br />
*Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.<br />
<br />
----<br />
----<br />
<br />
==='''''10-Agust-2009'''''===<br />
<br />
We made DNA purification from the Gel an we are planing start ligations this week.<br />
<br />
----<br />
----<br />
<br />
==='''''11-Agust-2009'''''===<br />
<br />
*Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations. <br />
<br />
----<br />
----<br />
<br />
==='''''12-Agust-2009'''''===<br />
<br />
We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).<br />
<br />
In this step we use 2 as backbone and 3 as insert, on the same 15 as backbone and 13 as insert, for 2-3 (BBa_K266000) we expect 3034 bp band as shown below, for 15-13 4074 bp. 4 as backbone 23 as insert. For ligation we use purifed DNA from agarosa Gel.<br />
<br />
<br />
<center><br />
{| border="0.5"<br />
! Mix Ligation <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 5μl<br />
|-<br />
|Insert DNA<br />
|align="right" | 25μl<br />
|-<br />
|T4 DNA Ligasa Buffer<br />
|align="right" | 3μl<br />
|-<br />
|T4 DNA ligasa <br />
|align="right" | 2μl<br />
|-<br />
|ATP<br />
|align="right" | 3μl<br />
|}<br />
</center><br />
<br />
The ligation left 17 hours overnight in 14ºC.<br />
<br />
----<br />
----<br />
<br />
==='''''13-Agust-2009'''''===<br />
<br />
We transform the ligation by thermic shock and plate.<br />
We received primers for 11 and 12.<br />
<br />
----<br />
----<br />
<br />
==='''''14-Agust-2009'''''===<br />
<br />
The transformation was successful we get colonies, we picket out for plasmid and left in 37ºC overnight.<br />
<br />
----<br />
----<br />
<br />
==='''''15-Agust-2009'''''===<br />
<br />
Only take off culture from camera and prepare for monday mini prep.<br />
<br />
----<br />
----<br />
<br />
==='''''17-Agust-2009'''''===<br />
<br />
We did mini prep and run out on a gel, we expect 3034 bp for L2_3(BBa_K266000) and 4074 bp for L15_13 (Bba_K266002) this are successful not so for 4_23 this is unsuccessful and proceed to repeat the ligation.<br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''18-Agust-2009'''''===<br />
<br />
The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight) <br />
<br />
----<br />
----<br />
<br />
==='''''19-Agust-2009'''''===<br />
<br />
Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.<br />
<br />
----<br />
----<br />
<br />
==='''''20-Agust-20009'''''===<br />
<br />
Purificated from gel for begin ligation.<br />
<br />
----<br />
----<br />
<br />
==='''''21-Agust-2009'''''===<br />
<br />
The transformation was through thermick shok and plate the weight we expect is amplicon 1515 bp 15_13 4074 bp total for Bba_K266003 5574 bp. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
----<br />
----<br />
<br />
==='''''22-Agust-2009'''''===<br />
<br />
The transformation was successful and we get colonies picket out for midi prep.<br />
For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.<br />
<br />
----<br />
----<br />
<br />
==='''''23-Agust-2009'''''===<br />
<br />
Meeting; we are detected a mistake on the primer, our primer musted include J23100 promotor now we have to do a extra ligation, and use J23100 as backbone.<br />
<br />
----<br />
----<br />
<br />
==='''''24-Agust-2009'''''===<br />
<br />
We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight<br />
<br />
----<br />
----<br />
<br />
==='''''25-Agust-2009'''''===<br />
<br />
Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation. <br />
<br />
"Check plasmid and gel picutre below"<br />
<br />
For ligation we going to do a mix and leave 17 hours in 14ºC camera.<br />
<br />
----<br />
----<br />
<br />
==='''''26-Agust-2009'''''===<br />
<br />
We transform by thermic shock and plate.<br />
<br />
----<br />
----<br />
<br />
==='''''27-Agust-2009'''''===<br />
<br />
The transformation was unsuccessful we haven't colonies.<br />
We going to repeat the ligation and left overnight incubation 14ºC.<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:23:12Z<p>Eniak: /* 31-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
After the 2009 catalog arrived we could resume the wetlab work because some of the biobricks we needed were not available on previous folders. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful, so we are now sure that the biobricks and methods we used work correctly so the next step is to extract the biobricks we are going to use for our project which are the following: <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we used the following method: <br />
<br />
*Elution: 3μl DNA from the well.<br />
*Put it on a 1.5 ml Eppendorf vial with competent cells.<br />
*Transformation was made by electroporation and recovery in LB media with Ampr for 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* E. coli Top10 cells which were transformed with biobricks F1610, K091146, K081016, K081009, R0051 and K081018 were plated and incubated for 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:19:08Z<p>Eniak: /* 29-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
After the 2009 catalog arrived we could resume the wetlab work because some of the biobricks we needed were not available on previous folders. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful, so we are now sure that the biobricks and methods we used work correctly so the next step is to extract the biobricks we are going to use for our project which are the following: <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we used the following method: <br />
<br />
*Elution: 3μl DNA from the well.<br />
*Put it on a 1.5 ml Eppendorf vial with competent cells.<br />
*Transformation was made by electroporation and recovery in LB media with Ampr for 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:14:41Z<p>Eniak: /* 29-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
After the 2009 catalog arrived we could resume the wetlab work because some of the biobricks we needed were not available on previous folders. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful, so we are now sure that the biobricks and methods we used work correctly so the next step is to extract the biobricks we are going to use for our project which are the following: <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:10:51Z<p>Eniak: /* 28-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
After the 2009 catalog arrived we could resume the wetlab work because some of the biobricks we needed were not available on previous folders. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart. <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:08:34Z<p>Eniak: /* 28-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
After the 2009 catalog arrived we could resume the wetlab work and take on the biobriks we need. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart. <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:01:59Z<p>Eniak: /* 10-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
With the 2009 catalog we retake the work and take on the biobriks we need. First of all we test them to be sure that the biobriks worked properly, for this we take a biobrik with RFP reporter (BBa_I3522), plate and incubate 17 hours in 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart. <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T07:00:19Z<p>Eniak: /* 09-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001. <br />
*Left for 16 hours at 37°C. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We don’t have transformed cells in this point we have to delay and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
With the 2009 catalog we retake the work and take on the biobriks we need. First of all we test them to be sure that the biobriks worked properly, for this we take a biobrik with RFP reporter (BBa_I3522), plate and incubate 17 hours in 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart. <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T06:58:32Z<p>Eniak: /* 08-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*We made the restrictions and put them on an agarose gel with 10 wells for 40 min.<br />
*We used 3μl DNA plus 2μl Buffer. <br />
*We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer <br />
*Run on only Plasmidic DNA but we have no DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, and take off DNA from the folder plating R0079, C0178, C0179, C0060, B0034, I739001 biobriks, <br />
*Left 16 hours in 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We don’t have transformed cells in this point we have to delay and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
With the 2009 catalog we retake the work and take on the biobriks we need. First of all we test them to be sure that the biobriks worked properly, for this we take a biobrik with RFP reporter (BBa_I3522), plate and incubate 17 hours in 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart. <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/CollaborationTeam:IPN-UNAM-Mexico/Collaboration2009-10-21T06:47:33Z<p>Eniak: /* 50pxLCG-UNAM-MEXICO & IPN-UNAM-MEXICO teams working together */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
<br />
<center><h1>Turing meets synthetic biology:</h1></center><br />
<br />
<center><h2>self-emerging patterns in an activator-inhibitor network</h2></center><br />
<br />
==[[Image:Month-icon.png | 50px]]LCG-UNAM-MEXICO & IPN-UNAM-MEXICO teams working together==<br />
<br />
When we heard that there was another iGEM team within our ''alma mater'' we started trying to contact them because we knew we could help each other to make better projects and produce better results.<br />
<br />
This is how we found [[Team:LCG-UNAM-Mexico| LCG-UNAM-Mexico iGEM team]] and since the early stages of our projects our team has been closely collaborating with them by exchanging ideas and discussing about experimental and theoretical issues we could have had in the future.<br />
<br />
Before we started to implement our projects we had some meetings where we finally met them. In this meetings we exposed to each other the general ideas we had and the way we were planning to model and implement them. We recieved a valuable advice from them on our project, specially on the way we were planning to regulate our system using IPTG and aTc inversors and we mentioned them some theoretical issues they cuold have on the modelling of their system, and we offered to help them. We also kept an active online communication with them and we were always curious to know what the other team was working on.<br />
<br />
When we were about to start implementing our system we had some bureaucratical issues on the customes with the current iGEM Kit Plate, and we needed some BioBricks of this Kit. Fortunately they already had it, so they kindly extracted, eluted, transformed, purified and sent them to us so we wouldn't lose more time. Certainly they saved us a lot of time, and we could started to work with a very little delay in comparison to the time we would have to wait for the arrival of the Kit. They also advised us in some technical details and tricks on the handling of the BioBricks, with good results in the wetlab.<br />
<br />
When they were modelling they found some difficulties on describing the dynamics of the diffussion component of their system, so we assesed them on what finally would be their cellular automaton with discrete diffusion. Since they had some troubles with a continuous model we proposed them to use a discrete version of what they were trying to do: the discrete Laplace operator, or well a partial differential equations system. Finally they chose to use a discrete version of Flick's law to solve this problem, with good results on their drylab.<br />
<br />
The collaboration between our team and LCG-UNAM was a very good idea, since it made things a lot easier for both teams and made possible to achieve better results. But definitely the best part of it was the creativity of our mutual contributions, since our teams together cover a very broad range of fields and we could complement each other very well. Two teams thinking together is better than one working alone.<br />
<br />
==[[Image:Month-icon.png | 50px]]Valencia survey==<br />
<br />
Our team help Valencia team answering some questions about synthetic biology. It was a great experience and an excelent idea from the members of Valencia team to gather this kind of information. We want to congratulate them for this iniciative.<br />
<br />
[[Image:V_IPNUNAMMexico.JPG |110px|center]]<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/Notebook/JulyTeam:IPN-UNAM-Mexico/Notebook/July2009-10-21T06:38:37Z<p>Eniak: /* 07-July-2009 */</p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
<br />
==='''''07-July-2009'''''===<br />
*Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).<br />
<br />
<center><br />
{| border="0.5"<br />
! Biobrick <br />
! Enzyme restriction<br />
|-<br />
|R0079<br />
|E, S<br />
|-<br />
|F1610<br />
|E, X<br />
|-<br />
|B0034<br />
|E, S<br />
|-<br />
|C0078<br />
|E, X<br />
|-<br />
|C0079<br />
|E, X<br />
|-<br />
|B0015<br />
|E, X<br />
|} <br />
</center><br />
<br />
<br />
*We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C. <br />
<br />
*Digest Mix:<br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - SpeI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme SpeI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H2O<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
<center><br />
{| border="0.5"<br />
! EcoRI - XbaI <br />
! Per reaction<br />
|-<br />
|Plasmidic DNA<br />
|align="right" | 3μl<br />
|-<br />
|Enzyme EcoRI<br />
|align="right" | 2μl<br />
|-<br />
|Enzyme XbaI<br />
|align="right" | 2μl<br />
|-<br />
|Buffer NBE<br />
|align="right" | 2μl<br />
|-<br />
|BSA<br />
|align="right" | 0.5μl<br />
|-<br />
|H20<br />
|align="right" | 10.5μl<br />
|-<br />
!Total<br />
|align="right" | 20μl<br />
|}<br />
</center><br />
<br />
----<br />
----<br />
<br />
==='''''08-July-2009'''''===<br />
<br />
*Carry out the restrictions on a 10 wells agarose gel 40 min.<br />
*3μl DNA 2μl Buffer. <br />
*We have not DNA on the Gels maybe the DNA volume is too low. We try again with 20μl DNA 3μl Buffer <br />
*Run on only Plasmidic DNA but we have not DNA. <br />
<br />
----<br />
----<br />
<br />
==='''''09-July-2009'''''===<br />
<br />
*Starting again, and take off DNA from the folder plating R0079, C0178, C0179, C0060, B0034, I739001 biobriks, <br />
*Left 16 hours in 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''10-July-2009'''''===<br />
<br />
We don’t have transformed cells in this point we have to delay and request 2009 catalog.<br />
----<br />
----<br />
<br />
==='''''28-July-2009'''''===<br />
<br />
With the 2009 catalog we retake the work and take on the biobriks we need. First of all we test them to be sure that the biobriks worked properly, for this we take a biobrik with RFP reporter (BBa_I3522), plate and incubate 17 hours in 37ºC. <br />
<br />
----<br />
----<br />
<br />
==='''''29-July-2009'''''=== <br />
<br />
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart. <br />
<br />
<center><br />
{| border="0.5"<br />
!Number<br />
!Biobrick<br />
|-<br />
|2<br />
|BBa_R0079<br />
|-<br />
|3<br />
|BBa_F1610<br />
|-<br />
|4<br />
|BBa_K091146<br />
|-<br />
|5<br />
|BBa_K093005<br />
|-<br />
|6<br />
|BBa_K081016<br />
|-<br />
|7<br />
|BBa_EC840<br />
|-<br />
|8<br />
|BBa_K081009<br />
|-<br />
|9<br />
|BBa_R0051<br />
|-<br />
|10<br />
|BBa_J06800<br />
|-<br />
|11<br />
|BBa_Q04121<br />
|-<br />
|12<br />
|BBa_B0034<br />
|-<br />
|13<br />
|BBa_C0079<br />
|-<br />
|14<br />
|BBa_C0179<br />
|-<br />
|15<br />
|BBa_B0015<br />
|-<br />
|16<br />
|BBa_K081018<br />
|-<br />
|17<br />
|BBa_K116640<br />
|}<br />
</center><br />
<br />
For transformation we use the same method as follow. <br />
<br />
*Elusion: 3μl DNA from the well.<br />
*Put it on a Eppendorf Vial 1.5 ml with competent cells.<br />
*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.<br />
*Plate on petri dishes and incubate 17 hours. <br />
<br />
----<br />
----<br />
<br />
==='''''31-July-2009'''''===<br />
<br />
* Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours<br />
<br />
----<br />
----<br />
<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniakhttp://2009.igem.org/Team:IPN-UNAM-Mexico/NotebookTeam:IPN-UNAM-Mexico/Notebook2009-10-21T06:30:52Z<p>Eniak: </p>
<hr />
<div>{{Template:IPN-UNAM-Mexico}}<br />
__FORCETOC__<br />
{{TOCright}} <br />
As summer proyect our experimental work began at july the 7th 2009 using 2008 bioparts catalog, unfortunately it didn't work properly, so we had a lot of trouble to take out DNA and transform into ''E. coli''. For this reason we had to delay lab work and request the 2009 catalog.<br />
<br />
<h1>[[Image:Month-icon.png | 50px]][[Team:IPN-UNAM-Mexico/Protocols|Protocols]]</h1><br />
<br />
<h2>[[Image:Month-icon.png | 50px]][[Team:IPN-UNAM-Mexico/Notebook/July|July]]</h2><br />
<h2>[[Image:Month-icon.png | 50px]][[Team:IPN-UNAM-Mexico/Notebook/August|August]]</h2><br />
<h2>[[Image:Month-icon.png | 50px]][[Team:IPN-UNAM-Mexico/Notebook/September|September]]</h2><br />
<h2>[[Image:Month-icon.png | 50px]][[Team:IPN-UNAM-Mexico/Notebook/October|October]]</h2><br />
Our synthetic construction consists of 29 bioparts, which needed 12 ligations and different strategies to make it functional as is described below:<br />
<br />
{{Template:IPN-UNAM-Mexico-footer}}</div>Eniak