Basic PCR Protocols (BioLine)

0.5 µL. Forward Primer (10 µM). 1 µL. 1 µL. 1 µL. Reverse Primer (10 µM). 1 µL. 1 µL. 1 µL. Taq DNA polymerase. 0.5 µL. 0.5 µL. 0.25 µL. DNA variable variable.
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Analysis of nucleic acids

Basic PCR Protocols (BioLine)

PCR MasterMix

Single Fly PCR

Fly genomic PCR

Colony/BAC PCR

Reagent

Volume

Volume

Volume

10X Buffer w\o MgCl2

2.5 µL

2.5 µL

2.5 µL

50 mM MgCl2

0.75§ µL

1.25** µL

0.75 µL

dNTP (10mM)

0.5 µL

0.5 µL

0.5 µL

Forward Primer (10 µM)

1 µL

1 µL

1 µL

Reverse Primer (10 µM)

1 µL

1 µL

1 µL

Taq DNA polymerase

0.5 µL

0.5 µL

0.25 µL

DNA

variable

variable

1-2.5 µL

ddH20

Up to 25 µL

Up to 25 µL

Up to 25 µL

Cycling conditions:

Temperature

Time

Initial denaturation

95°C

5-10 minutes

Denaturation

95°C

Annealing

45-65°C

1x

30 seconds ††

30 seconds

Elongation

72°C

~ 1-2 seconds / 25 bp

Final elongation

72°C

5 minutes

25-30 x

1x

§

1.5 mM final concentration Usually, 1.5 mM MgCl2 is the optimal concentration when dNTP are used at 200 µM. In this case, addition of MgCl2 is due to the presence of EDTA in the sample. †† Depends on primers melting temperature and template complexity.

**

Analysis of Nucleic Acids 7