Team:Technion/4_September_2012
Ilya
- Ran 5ul of the PCR products on a gel, got the expected bands.
- Purified the PCR products and measured concentration.
- Restriction digest of the pCP backbone, Fus2 and Fus2del inserts with XhoI and XbaI.
- Ran the pCP restriction products on a gel. The bands were very weak, which was unexpected because I took 3 ug of DNA. The nanodrop must have had an error when I measured the concentrations before the restriction.
- Gave all the miniprep products I have of pCP to Aya, she will concentrate it using the speed vac.
- Planned with Hila and Roee the set of reaction conditions for the assembly of two phage fragments. We will use 100, 250 and 400 bp overlaps with different incubation times with a T5 exonuclease in 37 degrees prior to the incubation with Gibson mix from NEB. In parallel, we will try using just the Gibson mix in the same reaction conditions (incubation in 37 degrees before the Gibson protocol).
Inbal
-mini prep for the BBa_B0015 biobrick , the concentrations are: 192.5, 252.5, 143.5 and 179 ng/ul (GREAT!).
-Digestion of BBa_B0015 with XbaI, after 2 hours at 37C I storaged the reactions at -20C.
-Also I digested the T7* RNAP with XbaI and SpeI, I cleaned the BBa_B0015 from all the buffers & enzymes by column cleaning kit, and the concentration I got was 56ng/ul (9ul in total).
Asaf
I made a fusion PCR between the RiboSwitch and the different polymerase genes with a Tm of 69C. It didn't work as you can see in the gel. Maybe the problem is with the Tm, so tomorrow I will try to find the right Tm using gradient PCR.
Hila
- CIP for the plasmid backbone.
- Cleanup for the plasmid backbone and the restriction reaction from yesterday.
- Overnight ligation to fragments 2C, 3C, 4C, 5C, 7C, 8C into the pSB1C3_MCS.
- Gel purification to fragments 1C and 6C.
Lior
Miniprep and nanodrop for: ALP + Sp6, K1F.
Noa
Evgeni
Shahar
After several attempts to produce NA & T3 Rnap failed, we tried a runing PCR with pPROLAR pproducts as templates.
we also did the reaction with 3 minutes elongation time. temprature for T3 : 58, 60, 62, 64. Temprature fore N4: 60, 62, 64 66. it worked with T3, but not with N4.
Rachel
After all the attempts to produce K1F RNAP with primers for pSB1A2 plasmid had failed, we had decided to try new approach: use K1F RNAP that had been produced with pPROLar primers as a template for PCR reaction in order to produce K1F RNAP suitable for cloning to pSB1A2 plasmid. So I ran PCR reaction, with 3 minutes elongation time (insted 1.5 as i did before), and 4 samples- each one with different Tm: 58C, 60C, 62C, 64C. This time, I got the desired bands (2700bp) at 60C and 62C!!!
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