From 2012.igem.org
Team:Technion/21_September_2012
Ilya
- Wiki, wiki, wiki....
Inbal
-Glycerol stocks for In1+ALP and In1+xyIE.
-Also, we have done the assay for the In1+ALP and In1+xyIE, but the results were the same as the control or much lesser than the control..... :/ at my opinion- I missed the eT7 RNAP somewhere in the cloning... Also, we checked the mCherry fluorescence, which is very high compare to the control- so I'm sure that the promoter with the mCherry is within the plasmid (pSB1AK3).
-I digested In1 with PstI and XmaI to verify if the clone is OK. After running on gel, I found out that the polymerase is missing in the part- Now I have to begin the cloning all over again!
- Restriction of pSB1C3 with XbaI- for my next steps of cloning the parts to the shipping plasmid.
-I made 10 starters for In1 from the transformation plates- I picked them randomly.. 1 starter for T7*RNAP+pSB1AK3 (that I know it works) and 4 starters of native T3 RNAP+pSB1AK3 from the transformation plates (picked by random).
-Colony PCR for 2 colonies of In1 part, I stored them at 16C at the end of the PCR.
Asaf
I got clones from all the transformations including my negative control. I descided due to lack of time
to proside to the next PCR and in doing so I will make sure if I got the right insert transformed.
I made starters from different clones and also streak plates.
Hila
- Transformation to Top10-Ca competent bacteria.
- Gibson assembly calibration using different T5 exonuclease concentrations, for the assembling of F4 and F5 together.
In this experiment we used three different fragments overlaps, in order to examine the T5 exo influence on the Gibson reaction.
The results are looking good‼!
We found that the T5 exo has a lot of influence on the reaction efficiency.
- Another PCR for fragments 1, 2, 3, and 8 with the 100bp fragments overlaps.
Lior
Noa
Evgeni
Shahar
Rachel
Restriction analyze with xbaI and speI in order to check if the ligation workd and was at the right direction. after digestion the pSB1AK3+RNAP (K1F/N4/T3), I ran the products on gel. I expected to receive two separate bends: one of 2800 bp (RNAP+terminator) and the second of 3100bp (pSB1AK3). As you can see in figure * we got separation only for two RNAPs: T7*(K17) and T7*(N4) one clone at each one , means the ligation works only for them.
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