Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 7.6.08

=7.7.08=

To Do:


 * Transform pKn003 and pKn004 into DH5&alpha;.
 * 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 DH5&alpha;.
 * 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.