Team:IPN-UNAM-Mexico/Notebook/August

From 2009.igem.org

(Difference between revisions)
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{{Template:IPN-UNAM-Mexico}}
{{Template:IPN-UNAM-Mexico}}
-
==='''''01-Agust-2009'''''===
+
==='''''03-Agust-2009'''''===
 +
 
 +
Resoults of previous transformations:
 +
 
 +
<center>
 +
{|
 +
|F1610
 +
|
 +
|Grow up
 +
|-
 +
|K091146
 +
|
 +
|Grow up
 +
|-
 +
|K081016
 +
|
 +
|Grow up
 +
|-
 +
|K081009
 +
|
 +
|Don't Grow
 +
|-
 +
|R0051
 +
|
 +
|Grow up
 +
|-
 +
|K081018
 +
|
 +
|Don't Grow
 +
|}
 +
</center>
 +
----
 +
----
 +
==='''''04-Agust-2009'''''===
Yesterday’s transformations were successful, continued to incubate it will ready for tomorrow’s mini prepped.  
Yesterday’s transformations were successful, continued to incubate it will ready for tomorrow’s mini prepped.  
Line 7: Line 40:
----
----
-
==='''''02-Agust-2009'''''===
+
==='''''05-Agust-2009'''''===
Line 32: Line 65:
|5
|5
|BBa_K093005
|BBa_K093005
-
|X &  
+
|X & P
|-
|-
|6
|6
Line 95: Line 128:
----
----
-
==='''''03-Agust-2009'''''===
+
==='''''06-Agust-2009'''''===
* Prepared falcon tubes with 5ml agar liquid Ampr  and inoculate.
* Prepared falcon tubes with 5ml agar liquid Ampr  and inoculate.
 +
*We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034)  we niknamed as AMPLICON.
----
----
----
----
-
==='''''04-Agust-2009'''''===
+
==='''''07-Agust-2009'''''===
-
*Miniprep next clones:  #16, #8, #23, #24, and prepare glycerol stocks.
+
*Miniprep next clones:  BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.
----
----
----
----
-
==='''''05-Agust-2009'''''===
+
==='''''10-Agust-2009'''''===
 +
 
 +
We made DNA purification from the Gel an we are planing start  ligations this week.
 +
 
 +
----
 +
----
 +
 
 +
==='''''11-Agust-2009'''''===
*Digest wit ECORI  we use this method to check out if is the plasmid correct and begin ligations.  
*Digest wit ECORI  we use this method to check out if is the plasmid correct and begin ligations.  
Line 116: Line 157:
----
----
-
==='''''08-Agust-2009'''''===
+
==='''''12-Agust-2009'''''===
-
We start with ligations 15-13 (BBa_K266002) and 2-3 (BBa_K266000).
+
We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).
-
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
+
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.
 +
 
 +
 
 +
<center>
 +
{| border="0.5"
 +
! Mix Ligation
 +
! Per reaction
 +
|-
 +
|Plasmidic DNA
 +
|align="right" | 5μl
 +
|-
 +
|Insert DNA
 +
|align="right" | 25μl
 +
|-
 +
|T4 DNA Ligasa Buffer
 +
|align="right" | 3μl
 +
|-
 +
|T4 DNA ligasa
 +
|align="right" | 2μl
 +
|-
 +
|ATP
 +
|align="right" | 3μl
 +
|}
 +
</center>
 +
 
 +
The ligation left 17 hours overnight in 14ºC.
----
----
----
----
-
==='''''09-Agust-2009'''''===
+
==='''''13-Agust-2009'''''===
-
"Check plasmid and gel picutre below"
+
We transform the ligation by thermic shock and plate.
 +
We received primers for 11 and 12.
-
We carried out ligation 4-23 (BBa_K2660059)  and  consider unsuccessful, we proceed to repeat the transformation.  
+
----
 +
----
 +
 
 +
==='''''14-Agust-2009'''''===
 +
 
 +
The transformation was successful we get colonys, we picket out for plasmid and left in 37ºC overnight.
----
----
----
----
-
==='''''11-Agust-2009'''''===
+
==='''''15-Agust-2009'''''===
-
We did ECORI  3 hours restriction for 3, L 4-23 (BBa_K266005), 17, 3, 7 we see an inespecific band on 17 maybe a sobredigestion, we proceede to repeat this one.  
+
Only take off culture from camera and prepare for monday mini prep.
 +
 
 +
----
 +
----
 +
 
 +
===''''17-Agust-2009'''''===
 +
 
 +
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.
"Check plasmid and gel picutre below"
"Check plasmid and gel picutre below"
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----
----
-
==='''''12 -Agust-2009'''''===
+
==='''''18-Agust-2009'''''===
-
We procede with 17_24 (Bba_K266001) ligation on this we going to use 17 as backbone and 24 as insert, we leave a 16 hours ligation in 14º camera.
+
The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight)
----
----
----
----
-
==='''''13-Agust-2009'''''===
+
===''''19-Agust-2009'''''===
-
We did  transformation using heat shock then plate.
+
Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.
----
----
----
----
-
==='''''14-Agust-2009'''''===
+
==='''''20-Agust-20009'''''===
-
The transformation was successful and proceed  to incubate tree  colonys 17 hours in liquid LB medium.
+
Purificated from gel for begin ligation.
----
----
----
----
-
==='''''15-Agust-2009'''''===
+
==='''''21-Agust-2009'''''===
-
We did midi prep for the ligation and carried out the plasmid is successful.
+
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.  
"Check plasmid and gel picutre below"
"Check plasmid and gel picutre below"
Line 173: Line 252:
----
----
-
==='''''17-Agust-2009'''''===
+
==='''''22-Agust-2009'''''===
-
We going to proceed with J23100 – 11 (Bba_K266008) J23100 is a constitutive promoter and we going to use as backbone 11 as insert. We leave overnight ligation in 14º camera.
+
The transformation was successful and we get colonys picket out for midi prep.
-
 
+
For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.
-
"Check plasmid and gel picutre below"
+
----
----
----
----
-
==='''''18-Agust-2009'''''===
+
==='''''23-Agust-2009'''''===
-
We did a transformation for thermic shock and plate, we expect colonys for midi
+
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.
----
----
----
----
-
==='''''19-Agust-2009'''''===
+
==='''''24-Agust-2009'''''===
-
The transformation was unsuccessful we dont get colonys we going to try again.
+
We going to do double digest for J23100  ECORI and SPEI and 11 like insert 17 hours digest overnight
----
----
----
----
-
==='''''20-Agust-2009.'''''===
+
==='''''25-Agust-2009'''''===
-
We get Amplicon – L15-13  (BBa_K266003): Restriction digest 16 hours  at 14º (overnight)  then  we will use thermic shock for cells transform.  
+
Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation.  
-
L 2-3 (BBa_K266000) we’re doing ECORI  3 hours digest at 37º.
+
"Check plasmid and gel picutre below"
-
For BBa P1010 we take off the ccdB gene for this use PSTI and ECORI double restriction we going to use this part as chloramphenicol resistance vector .  
+
For ligation we going to do a mix and leave 17 hours in 14º camera.
 +
 
 +
----
 +
----
 +
 
 +
==='''''26-Agust-2009''''
 +
 
 +
We transform by thermic shock and plate.
 +
 
 +
----
 +
----
-
For 24-17 BBa_K266001 we get this ligation and proceed to double digest
+
==='''''27-Agust-2009'''''===
 +
The transformation was unsuccessful we haven't colonies.
 +
We going to repeat the ligation and left overnight incubation 14ºC.
{{Template:IPN-UNAM-Mexico-footer}}
{{Template:IPN-UNAM-Mexico-footer}}

Revision as of 06:19, 21 October 2009


BannerUNAM.jpg

03-Agust-2009

Resoults of previous transformations:

F1610 Grow up
K091146 Grow up
K081016 Grow up
K081009 Don't Grow
R0051 Grow up
K081018 Don't Grow

04-Agust-2009

Yesterday’s transformations were successful, continued to incubate it will ready for tomorrow’s mini prepped.

05-Agust-2009

We made mini preped (protocol) and did the follow restrictions:

Number Biobrick Restriction
2 BBa_R0079 S & P
3 BBa_F1610 X & P
4 BBa_K091146 S & P
5 BBa_K093005 X & P
6 BBa_K081016 X & P
7 BBa_EC840 X & P
8 BBa_K081009 X & P
9 BBa_R0051 S &P
10 BBa_J06800 X & P
11 BBa_Q04121 X & P
12 BBa_B0034 S & P
13 BBa_C0079 E & S
14 BBa_C0179 E & S
15 BBa_B0015 E & X
16 BBa_K081018 E & S
17 BBa_K116640 X & P
23 BBa_S03154 X & P
24 BBa_k145201 E & S

06-Agust-2009

  • Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.
  • We designed primers for 11 (Bba_Q04121) & 12 (BBa_B0034) we niknamed as AMPLICON.

07-Agust-2009

  • Miniprep next clones: BBa_K081018, BBa_K081009, Bba_S03154, BBa_K145201, and prepare glycerol stocks.

10-Agust-2009

We made DNA purification from the Gel an we are planing start ligations this week.

11-Agust-2009

  • Digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations.

12-Agust-2009

We start with ligations 15-13 (BBa_K266002), 2-3 (BBa_K266000) and 4_23 (BBa_K266005).

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.


Mix Ligation Per reaction
Plasmidic DNA 5μl
Insert DNA 25μl
T4 DNA Ligasa Buffer 3μl
T4 DNA ligasa 2μl
ATP 3μl

The ligation left 17 hours overnight in 14ºC.

13-Agust-2009

We transform the ligation by thermic shock and plate. We received primers for 11 and 12.

14-Agust-2009

The transformation was successful we get colonys, we picket out for plasmid and left in 37ºC overnight.

15-Agust-2009

Only take off culture from camera and prepare for monday mini prep.

'17-Agust-2009

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.

"Check plasmid and gel picutre below"

18-Agust-2009

The next step is make a ligation Amplicon – 15_13 (Bba_K266003) from the 15_13 DNA purification we left 17 hours ligation (overnight)

'19-Agust-2009

Do double digestion for 15_13 as insert and aplicon as backbone overnight digest.

20-Agust-20009

Purificated from gel for begin ligation.

21-Agust-2009

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.

"Check plasmid and gel picutre below"

22-Agust-2009

The transformation was successful and we get colonys picket out for midi prep. For ligations we're use midi in this step we need more DNA for ligations. The plasmid weight grow up.

23-Agust-2009

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.

24-Agust-2009

We going to do double digest for J23100 ECORI and SPEI and 11 like insert 17 hours digest overnight

25-Agust-2009

Carried out on the digest on a gel and we watch correct weight we going to proceed to ligation.

"Check plasmid and gel picutre below"

For ligation we going to do a mix and leave 17 hours in 14º camera.

===26-Agust-2009'

We transform by thermic shock and plate.

27-Agust-2009

The transformation was unsuccessful we haven't colonies. We going to repeat the ligation and left overnight incubation 14ºC.


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