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

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Contents

HotStar PCR

Rxn Mix

Reagent Volume/Amount
Paeru Genomic DNA Template0.5 μl
HotStar Polymerase1.0 μl
BT27361 μl 50 μM Stock
BT27371 μl 50 μM Stock
5X Buffer 10 μl
Mg2+None for now, may optimize later
5X Q-Solution10 μl
dH2026.5 μl
Total50 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 Denaturation95oC5 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation94oC15 Seconds
Cyclic Annealing50oC1 minute
Cyclic Extension72oC1 minute
Repeat Cyclic Steps 35x
Final Extension72oC10 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

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