Matt Gethers/CRI, Thailand/Labwork/PCRs/Amplification of HmgA Downstream Fragment
Amplification of HmgA Downstream Fragment
|Genomic DNA Template||0.5 μl from So Pa Pan stock|
|Primer 1033||4 μl 1 μM Stock|
|Primer 2724||4 μl 1 μM Stock|
|dNTPs||1 μl 10 mM Stock|
|Buffer S||5 μl|
1033: CGC TAC GAG GAC AGC CGC
2724: CTG GTC GTT GTC GTC GTA GA
Annealing Temperature: 55.0oC
Extension Time: 3 minutes (2 minutes/Kb) Cycle
|Initial Denaturation||95oC||3 minutes|
|Cyclic Denaturation||94oC||40 Seconds|
|Cyclic Annealing||55oC||40 Seconds||Unsure of best temp|
|Cyclic Extension||72oC||3 minutes|
|Repeat Cyclic Steps 35x|
|Final Extension||72oC||10 minutes|
Didn't include 7 minute 72oC extension at end; suspect the 2 minute 95oC step at the start might not have launched (failed to save?). Used 51.5oC annealing temp and 6 minute extension time.
Ensured that there is a 2 minute 95oC denaturation step before the cycle (which includes a 94oC denaturation step), changed the annealing temp to 55oC, changed the extension time to 2 minutes (between 1.5 and 2 minutes/Kb).
Made the following changes from the 6.13.08 evening run:
- 95oC initial step 3 minutes (up from 2)
- 95oC cyclic step 40 seconds (up from 94, 30)
- 55oC cyclic step 40 seconds (up from 30)
- 72oC cyclic step 3 minutes (up from 2)
- 72oC final step 10 minutes (up from 7 minutes)
- Used Buffer S (has Mg2+) rather than Buffer A.
- Made 1 μM aliquots of primers 1032, 1033, and 2724; used 2 μl of 1033 in each reaction and 4 μl of 1032 and 2724 in each reaction (oops, that's 2x what it should be).
- Made only 25 μl reactions this time around (just to see if the PCR works this time).
The most recent protocol worked, so I'm making another batch this morning alongside the HmgR ORF (both have the same annealing temp, so I don't even have to use the gradient feature).