Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 7.6.08
7.7.08
To Do:
- Transform pKn003 and pKn004 into DH5α.
- Prepare colony PCR Protocols for putative transformants.
- Align sequence data for pKn001 and pKn002 when they arrive.
Summary:
Went home a bit sick today. Postponed everything until tomorrow.
7.8.08
To Do:
- Transform pKn003 and pKn004 into DH5α.
- Prepare colony PCR Protocols for putative transformants.
- Align sequence data for pKn001 and pKn002 when they arrive.
Summary:
I ran the transformation without problems. Will get the plates tomorrow and run colony PCRs.
7.9.08
To Do:
- Prepare colony PCR Protocols for putative transformants.
- Make colony suspensions and run PCR.
- Align sequence data for pKn001 and pKn002 when they arrive.
Summary:
I wrote the colony PCR protocols to assay pKn003 transformants and pKn004 transformants. I also prepared the colony suspensions and PCRs. Will run gel tomorrow.
7.10.08
To Do:
- Run out PCR products on a gel, look for correct products.
- If correct products show up, inoculate cultures for prep and glycerol.
- If no correct products show up, either screen more or prepare for another transformation with controls.
- Align sequence data for pKn001 and pKn002 when they arrive.
Summary:
I ran out the products on a gel. I think I accidentally used pKn002 in the pKn003 construction and it seems my digest or purification of the SphI-HindIII cut pKn002 is inefficient in the construction of pKn004. I need to try these steps again, so I've digested pKn002 with SphI-HindIII again and ran on a PCR clean up column to get rid of the 10 bp fragment (I hoping it's a purification issue and not a digestion issue). I will then ligate the HmgR downstream fragment (p'So #4) into it again on Monday. I'm just going to ligate HmgA upstream fragment (p'So #2) into pKn001 again seeing as how I think I used the wrong tubes, so I digested pKn001 with HincII and used the End-It kit.
