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Team:IPN-UNAM-Mexico/Notebook
From 2009.igem.org
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:
June
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
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
September
October
