Matt Gethers/CRI, Thailand/Labwork/PCRs/Screening for Presence and Directionality of HmgA Upstream Fragment in pUC18

=Screening for Presence and Directionality of HmgA Upstream Fragment in pUC18 (pKn003)=

Rxn Conditions
Annealing Temperature: 55oC (2.7 degrees below BT1188)

Extension Time: 1 minute 40 seconds (1504 bp at 1 min/kb ~ 1 minute 30 seconds)


 * Note: This PCR will fail entirely if the insert hasn't been ligated into the vector as the BT1188 binding site is on the HmgA upstream fragment.

Cycle (Taq)

7.9.08
I ran the protocol using 12 10 &mu;l reactions (6 for pKn003, 6 for pKn004); 9 &mu;l PCR mix and 1 &mu;l sample. Ran protocol as written except the extension time was 1 minute 50 seconds so the PCR could be done jointly with the pKn004 colony PCR. I got a weird result - product of the wrong length where I shouldn't have anything at all if the insert failed to ligate into the vector. Because the product length is the exact same as the failed pKn004 ligation (which gives a product even if the insert failed to ligate into the vector), I think I might have just mixed up the tubes during the ligation. Gel results are here.

7.15.08
I ran the protocol using 15 10 &mu;l reactions (7 for pKn003, 8 for pKn004); 9 &mu;l PCR mix and 1 &mu;l sample. Ran protocol as written except the extension time was 1 minute 50 seconds so the PCR could be done jointly with the pKn004 colony PCR. I still got a weird result - product of the wrong length where I shouldn't have anything at all if the insert failed to ligate into the vector. However, I'm certain I used the right vector for ligation this time and I got one colony (colony 5) that sequenced at almost the right length (looks like 1200, I was expecting 1500). I'm going to make a glycerol and send for sequencing. Gel results are here.

7.21.08
I screened 12 more colonies from the transformation last week of pKn003.L2. Found that colony #7 gave a good product. Note that I'm make notes after the fact (8.13.08) and that I forgot to record anything at the time.