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DNA > POLYMERASE CHAIN REACTION (PCR)

> Polymerase Chain Reaction

    > Melting Temperature Calculation

 

Polymerase Chain Reaction

Get a printable version of this protocol (requires Adobe Acrobat

For normal PCR, we have found that the following conditions appear to work well.

Dilutions:

    Primers diluted to 25pmol/uL (1:60 of 300pmol ul-1 stock)
    Template plasmid DNA diluted to 100pg/uL (2 serial dilutions of 1/100 of stock)
    dNTPs prepared at 5 mM stock

Set up the following reactions

 

100uL Reaction

template

2

primer 1

1

primer 2

1

10X buffer

10

dNTPs

4

H2O

81

Vent

1

Sample PCR conditions
  1. First, 1 cycle for 1 min at 94 C
  2. Then, 25 cycles:
1 min in 94C
1 min in melting temperature
1 min/kbase at 72C
72C 2 min
hold 4C

For DNA stretches of up to 1kbp, then 30" for each step in the cycle is usually enough.

For complete (3-4kbp) plasmids, increase the extension time to 1'30"

Calculating Primer Concentration

C= A x10(6) /E

Where:

C= amount of DNA primer in pmoles
A= absorbance at 260nm
E= extinction coefficients of each nucleotide

(11.7x #G's) + (7.3x #C's) + (15.4x #A's) + (8.8x #T's)

Volume to resuspend primer in to get 25 pmol/uL = c/25