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

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

Rxn Mix

Reagent Volume/Amount
Paeru Genomic DNA Template 0.5 μl
Taq 0.5 μl
M13 for 4 μl 1 μM Stock
BT1188 4 μl 1 μM Stock
dNTPs 1 μl 10 mM Stock
DMSO 2.5 μl
Buffer 5 μl
Mg2+ 2.5 μl
dH20 30 μl
Total 50 μl

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)

Step Temperature Duration Notes
Initial Denaturation 95oC 2 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation 94oC 30 Seconds
Cyclic Annealing 55oC 30 Seconds Unsure of best temp
Cyclic Extension 72oC 1 minute 40 seconds
Repeat Cyclic Steps 35x
Final Extension 72oC 10 minutes

Run Notes

7.9.08

I ran the protocol using 12 10 μl reactions (6 for pKn003, 6 for pKn004); 9 μl PCR mix and 1 μl 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 μl reactions (7 for pKn003, 8 for pKn004); 9 μl PCR mix and 1 μl 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.