Team:BYU Provo/Team Thermosensor/Week12
Week 12 (Jul 3 - 9)
Removed plates from incubator
Plated 8 colonies onto a “pie plate” to check for the amp resistance. Since plate T36-1 was left for longer than just overnight it might not be truly antibiotic resistant (have the plasmid transformed) so we’ll take those 8 colonies and check by incubating overnight on plate T36-2 - Mark
I talked to Dr. Grose about the results from the last colony PCR (on pg. 33). She suggested we redo the colony PCR from plates ∆T30-1 and ∆T30-2 to make sure we didn’t mix things up because the lower bands look like they could have been lacZ and then we would need to redo GFP. So let’s redo the colony PCR from plates ∆T30-1 and ∆T30-2 using the same protocol Devin used on page 32. I don’t have time today, but I can do that a little later this week if no one else can.
Colony PCR for pPLATBad/lacZ-1 (∆T30-1) T37-1
- Taq DNA polymerase added las to master mix
- IG40 and IG12 as primers
Colony PCR for pPLATBad/lacZ-1 (T36-2) T37-2
- Taq added last
- IG 40 and IG12 as primers
Colony PCR for pPLATBad/GFP (∆T30-2) T37-3
- IG26 and IG12 as primers. Taq added last. - Mark
- Lanes 2, 4, and 5 look great -going to set up overnights. -Mark
- Lane 3 was a neg. control because there was no growth from that colony.
Just to be clear: colonies 1,3 will be “overnighted” in LB-amp from plate T36-2
- T38-1 and 2: from colony 1
- T38-3 and 4: from colony 2
Colony PCR 37-3 (from plate ∆T30-2) pPLATBad-GFP
- These are at about 300 bp - a little small for our GFP insert. Dr. Grose suggested we purify them anyway and sequence to see what’s going on.
Colonies 2 and 3 will be overnighted to see what’s going on.
- T38-5 and 6: from colony 2 from plate ∆T30-2
- T38-7 and 8: from colony 3 from ∆T30-2
pIG15: pLATBADlacZ colony 1 (plate T36-2)
pIG16: pLATBADlacZ colony 3 (plate T36-2)
pIG17: pLATBADGFP colony 2 (plate ∆T30-2)
pIG18: pLATBADGFP colony 2 (plate ∆T30-2)
Next step is to digest thermosensor and pIG16 and get on to the ligation! -Mark and Chet