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

From OpenWetWare
Jump to: navigation, search

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: 55.0oC

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 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.

6.13.08 Evening

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).

6.16.08

Made the following changes from the 6.13.08 evening run:

Protocol:

  • 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)

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).