Matt Gethers/CRI, Thailand/Labwork/PCRs/Amplification of HmgA Downstream Fragment

Amplification of HmgA Downstream Fragment

Rxn Mix

Reagent	Volume/Amount
Genomic DNA Template	0.5 μl from So Pa Pan stock
Pfu	0.5 μl
Primer 1033	4 μl 1 μM Stock
Primer 2724	4 μl 1 μM Stock
dNTPs	1 μl 10 mM Stock
DMSO	2.5 μl
Buffer S	5 μl
dH20	32.5 μl
Total	50 μl

Primers

1033: CGC TAC GAG GAC AGC CGC

2724: CTG GTC GTT GTC GTC GTA GA

Rxn Conditions

Annealing Temperature: 55.0^oC

Extension Time: 3 minutes (2 minutes/Kb) Cycle

Step	Temperature	Duration	Notes
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

Run Notes

6.13.08 Morning

Didn't include 7 minute 72^oC extension at end; suspect the 2 minute 95^oC step at the start might not have launched (failed to save?). Used 51.5^oC annealing temp and 6 minute extension time.

6.13.08 Evening

Ensured that there is a 2 minute 95^oC denaturation step before the cycle (which includes a 94^oC denaturation step), changed the annealing temp to 55^oC, changed the extension time to 2 minutes (between 1.5 and 2 minutes/Kb).

6.16.08

Made the following changes from the 6.13.08 evening run:

Protocol:

• 95^oC initial step 3 minutes (up from 2)
• 95^oC cyclic step 40 seconds (up from 94, 30)
• 55^oC cyclic step 40 seconds (up from 30)
• 72^oC cyclic step 3 minutes (up from 2)
• 72^oC final step 10 minutes (up from 7 minutes)

Reagents:

• 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).

6.17.08

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).