DNA	> POLYMERASE CHAIN REACTION (PCR)	> Polymerase Chain Reaction
		> Melting Temperature Calculation

 

Polymerase Chain Reaction

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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 94º C
2. Then, 25 cycles:

	1 min in 94°C
	1 min in melting temperature
	1 min/kbase at 72°C
	72ºC 2 min
	hold 4°C 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