Team:IPN-UNAM-Mexico/Notebook

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(Difference between revisions)
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We plated  Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and get incube 17 hours
We plated  Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and get incube 17 hours
 +
----
 +
----
<h1>[[Image:Month-icon.png | 50px]] August </h1>
<h1>[[Image:Month-icon.png | 50px]] August </h1>
 +
 +
==='''''01-Agust-2009'''''===
 +
 +
Yesterday’s transformations were successful, continued to incubate it will ready for tomorrow’s mini prepped.
 +
 +
----
 +
----
 +
 +
 +
==='''''02-Agust-2009'''''===
 +
 +
 +
We made mini preped (protocol) and did the follow restrictions.
 +
 +
{| border="0.5"
 +
!Number
 +
!Biobrick
 +
!Restriction
 +
|-
 +
|2
 +
|BBa_R0079
 +
|S & P
 +
|-
 +
|3
 +
|BBa_F1610
 +
|X & P
 +
|-
 +
|4
 +
|BBa_K091146
 +
|S & P
 +
|-
 +
|5
 +
|BBa_K093005
 +
|X &
 +
|-
 +
|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
 +
|}
 +
 +
----
 +
----
 +
 +
==='''''03-Agust-2009'''''===
 +
 +
We prepare falcon tubes with 5ml agar liquid Ampr  and inoculate colonys.
 +
 +
----
 +
----
 +
 +
==='''''04-Agust-2009'''''===
 +
 +
We did miniprep next clones:  #16, #8, #23, #24, and prepare glycerol stocks.
 +
 +
----
 +
----
 +
 +
==='''''05-Agust-2009'''''===
 +
 +
We made digest wit ECORI  we use this method to check out if is the plasmid correct and begin ligations.
 +
 +
----
 +
----
 +
 +
==='''''08-Agust-2009'''''===
 +
 +
We start with ligations 15-13 (BBa_K266002) and 2-3 (BBa_K266000)
 +
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
 +
 +
----
 +
----
 +
 +
==='''''09-Agust-2009'''''===
 +
 +
"Check plasmid and gel picutre below"
 +
 +
We carried out ligation 4-23 (BBa_K2660059)  and  consider unsuccessful, we proceed to repeat the transformation.
 +
 +
----
 +
----
 +
 +
==='''''11-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.
 +
 +
"Check plasmid and gel picutre below"
 +
 +
----
 +
----
 +
 +
==='''''12 -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.
 +
 +
----
 +
----
 +
 +
==='''''13-Agust-2009'''''===
 +
 +
We did  transformation using thermic shock then plate. 
 +
 +
----
 +
----
 +
 +
==='''''14-Agust-2009'''''===
 +
 +
The transformation was successful and proceed  to incubate tree  colonys 17 hours in liquid LB medium.
 +
 +
----
 +
----
 +
 +
==='''''15-Agust-2009'''''===
 +
 +
We did midi prep for the ligation and carried out the plasmid is successful.
 +
 +
"Check plasmid and gel picutre below"
 +
 +
----
 +
----
 +
 +
==='''''17-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. 
 +
 +
"Check plasmid and gel picutre below"
 +
 +
----
 +
----
 +
 +
==='''''18-Agust-2009'''''===
 +
 +
We did a transformation for thermic shock and plate, we expect colonys for midi
 +
 +
----
 +
----
 +
 +
==='''''19-Agust-2009'''''===
 +
 +
The transformation was unsuccessful we dont get colonys we going to try again.
 +
 +
----
 +
----
 +
 +
==='''''20-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.
 +
 +
L 2-3 (BBa_K266000) we’re doing ECORI  3 hours digest at 37º.
 +
 +
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 24-17 BBa_K266001 we get this ligation and proceed to double digest
 +
 +
<h1>[[Image:Month-icon.png | 50px]] September </h1>
<h1>[[Image:Month-icon.png | 50px]] September </h1>

Revision as of 17:53, 20 October 2009


BannerUNAM.jpg

As summer proyect our experimental work began at 07 - july - 2009 using 2008 bioparts catalog, unfurtunly it dosen’t work properly, we had to much troubles to take out DNA and transform, E. Coli for this we had to delay and request 2009 catalog.

Our proyect uses 29 bioparts, 12 ligations and diferent strategys to make it functional as follow:

Month-icon.png June

Month-icon.png July

07-July-2009

We digest the follow biobriks with ECORI (E), SPEI (S), and XBAI (X).

Biobrick Enzime restriction
R0079 E, S
F1610 E, X
B0034 E, S
C0078 E, X
C0079 E, X
B0015 E, X


We propose this restrictions for standar assembly for the digest we use a 17 hours incubation at 37º camera. Digest Mix:


ERCO RI - SPEI Per reaction
Plasmidic DNA 3μl
Enzima ECORI 2μl
Enzima SPEI 2μl
Buffer NBE 2μl
BSA 0.5μl
H2O 10.5μl
Total 20μl


ECORI - XBAI Per reaction
Plasmidic DNA 3μl
Enzima ECORI 2μl
Enzima XBAI 2μl
Buffer NBE 2μl
BSA 0.5μl
H20 10.5μl
Total 20μl

08-July-2009

We carried out the restrictions on a 10 wells agarosa gel 40 min. 3μl DNA 2μl Buffer. We have not DNA on the Gels maybe the DNA volume is so low. We try again with 20μl DNA 3μl Buffer We run on only Plasmidic DNA but we have not DNA.

09-July-2009

We start from cero, and take off DNA from the folder plateing R0079, C0178, C0179, C0060, B0034, I739001 biobriks, We left 16 hours in 37º camera.

10-July-2009

We don’t have transform cells in this point we have to delay and request 2009 catalog.

28-July-2009

With the 2009 catalog we retake the work and take on the biobriks we need. First of all we did a test to be sure that the biobriks worked properly for this we take a biobrik with RFP reporter (BBa_I3522) and plate, we incubate 17 hours in 37º camera.

29-July-2009

The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart.


Number Biobrick
2 BBa_R0079
3 BBa_F1610
4 BBa_K091146
5 BBa_K093005
6 BBa_K081016
7 BBa_EC840
8 BBa_K081009
9 BBa_R0051
10 BBa_J06800
11 BBa_Q04121
12 BBa_B0034
13 BBa_C0079
14 BBa_C0179
15 BBa_B0015
16 BBa_K081018
17 BBa_K116640

For transformation we use the same method as follow.

Elusion: 3μl DNA from the well, and get in Eppendorf 1.5 ml with competent cells. We use electoporation shock to transform and recovery in LB ampr 1 hour and a half then we plate on petri dishes and get incubate 17 hours.

31-July-2009

We plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and get incube 17 hours

Month-icon.png August

01-Agust-2009

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


02-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 &
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

03-Agust-2009

We prepare falcon tubes with 5ml agar liquid Ampr and inoculate colonys.

04-Agust-2009

We did miniprep next clones: #16, #8, #23, #24, and prepare glycerol stocks.

05-Agust-2009

We made digest wit ECORI we use this method to check out if is the plasmid correct and begin ligations.

08-Agust-2009

We start with ligations 15-13 (BBa_K266002) and 2-3 (BBa_K266000) 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

09-Agust-2009

"Check plasmid and gel picutre below"

We carried out ligation 4-23 (BBa_K2660059) and consider unsuccessful, we proceed to repeat the transformation.

11-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.

"Check plasmid and gel picutre below"

12 -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.

13-Agust-2009

We did transformation using thermic shock then plate.

14-Agust-2009

The transformation was successful and proceed to incubate tree colonys 17 hours in liquid LB medium.

15-Agust-2009

We did midi prep for the ligation and carried out the plasmid is successful.

"Check plasmid and gel picutre below"

17-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.

"Check plasmid and gel picutre below"

18-Agust-2009

We did a transformation for thermic shock and plate, we expect colonys for midi

19-Agust-2009

The transformation was unsuccessful we dont get colonys we going to try again.

20-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.

L 2-3 (BBa_K266000) we’re doing ECORI 3 hours digest at 37º.

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 24-17 BBa_K266001 we get this ligation and proceed to double digest


Month-icon.png September

Month-icon.png October


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