Matt Gethers/CRI, Thailand/Labwork/PCRs/Amplification of HmgR ORF using HotStar

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HotStar PCR

Rxn Mix

Reagent Volume/Amount
Paeru Genomic DNA Template 0.5 μl
HotStar Polymerase 1.0 μl
BT2736 1 μl 50 μM Stock
BT2737 1 μl 50 μM Stock
5X Buffer 10 μl
Mg2+ None for now, may optimize later
5X Q-Solution 10 μl
dH20 26.5 μl
Total 50 μl

Rxn Conditions

Annealing Temperature: 50oC (5 degrees below 2736 annealing temp)

Extension Time: 1 minute (1 min/kb, 804 bases)

Cycle

Step Temperature Duration Notes
Initial Denaturation 95oC 5 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation 94oC 15 Seconds
Cyclic Annealing 50oC 1 minute
Cyclic Extension 72oC 1 minute
Repeat Cyclic Steps 35x
Final Extension 72oC 10 minutes

Run Notes

7.23.08

I ran two PCRs to amplify the HmgR ORF: one with Buffer Q and the other without. I made a master mix, then aliquotted 40 μl to each of two tubes. I then added 10 μl Buffer Q to one and the same volume of water to the other. Used the reaction conditions as written. Gel results here.

8.2.08

I ran one PCR to amplify the HmgR ORF with buffer Q. I just made the reaction as in the protocol and ran with the conditions written above.

References

HotStar HiFidelity PCR Handbook.pdf