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Team:IPN-UNAM-Mexico/Notebook/August
From 2009.igem.org
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
- Prepared falcon tubes with 5ml agar liquid Ampr and inoculate.
04-Agust-2009
- Miniprep next clones: #16, #8, #23, #24, and prepare glycerol stocks.
05-Agust-2009
- 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 heat 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
