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Table 2 Second-round PCR conditions

From: Affordable drug resistance genotyping of HIV-1 reverse transcriptase, protease and integrase genes, for resource limited settings

Second-round PCR Mastermix

Reagent

Volume per reaction (µl)

Concentration per reaction

Nuclease-free Water

18.4

–

10X PCR Buffer

2.5

1X

MgCl2 (50 mM)

1.0

2 mM

dNTP (10 mM)

0.5

0.2 mM

Pro1 (5 µM)

0.25

0.05 µM

5066R (5 µM)

0.25

0.05 µM

Platinum Taq DNA Polymerase

0.1

–

Total volume

23

–

Thermocycling conditions

 

Temperature (oC)

Time

Cycle(s)

Pre-denaturation

94

2 min

1

Denaturation

95

10 s

40

Annealing

56

20 s

Extension

72

2 min

Final extension

72

10 min

1

Hold

4

∞

Hold

  1. dNTP deoxynucleoside triphosphate; MgCl2 magnesium chloride; mM millimolar; µl microliter; µM micromolar; oC Degrees Celsius
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AIDS Research and Therapy

ISSN: 1742-6405

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