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PCR Reactions

Prepare the following reactions in a PCR tube:

If you are using the 2x Taq Master Mix:

Component

25 μl reaction

50 μl reaction

Final Concentration

2X Taq Master Mix(add last)

12.5 µl

25 μl

1X

10 µM Forward Primer

0.5 µl

1 μl

0.2 µM (0.05–1 µM)

10 µM Reverse Primer

0.5 µl

1 μl

0.2 µM (0.05–1 µM)

Template DNA

~50ng

~100ng

<1,000 ng

Nuclease-free water

~10 µl

~20 µl

 

 

 

If you are using another enzyme not in a Master MIx:

Component

25 μl reaction

50 μl reaction

Final Concentration

10X Polymerase/PCR Buffer

2.5 µl

5 μl

1X

10 mM dNTPs

0.5 µl

1 μl

200 µM

10 µM Forward Primer

0.5 µl

1 μl

0.2 µM (0.05–1 µM)

10 µM Reverse Primer

0.5 µl

1 μl

0.2 µM (0.05–1 µM)

Template DNA

~50ng

~100ng

<1,000 ng

Taq DNA Polymerase*

0.5 µl

1 µl

1.25 units/50 µl PCR

Nuclease-free water

to 25 µl

to 50 µl

 

 

 

Gently mix the reaction and spin down in microcentrifuge.

Cycling Conditions for a Routine PCR:

STEP

TEMP

TIME

Initial Denaturation

95°C

30 seconds

30 Cycles     Melting Temp.

                    Annealing Temp.

                    Extension Temp.

95°C

56°C

68°C

30 seconds

30 seconds

1 minute/kb

Final Extension

68°C

8 minutes

Hold

4-10°C

 

 

I always use 56 or 56.5C for my Annealing Temperature unless the primer has a large region of random nucleotides, then I might drop it down to 52-54C. This has worked on 99.99% of all of my PCR reactions.

 

 

Modified from NEB: https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction