PCR base cloning -Primer Selection

Last update April 5, 2008

Here in is an example of PCR based cloning of gene STX4 to to pET 20 plasmid vector. pET 20 vector is an C-terminal 6xHis-tag protein expression vector, which is available from "Novagene". If you want to know more about this protein expression system read here.

Copy and Paste the Reverse primer here .

5' 3'

Example Primer Selection


Enter your sequence here :


Length bp
GC content %
Molecular Weight: g/mol
Melting Temperature (Tm) °C
Epsilon 260 nm
Quantity of 1 OD is equal to nmoles µg

There are a number of different ways to calculate the melting temperature of an oligonucleotide. All of these methods will give different results. Please keep in mind that all of these calculations are theoretical! Exact melting temperatures must be determined empirically. For oligonucleotides <= 20 bases, use the classical Tm = 2(A + T) + 4(G + C) calculation for melting temperature, according to Wallace rule for short oligonuceotides (1). For oligonucleotide > 20 bases, calculation is based on the Nearest-Neighbor formula with [salt] = 50 mM and single strand concentration = 250 pM (2). You can find more comprehensive calculation of oligos melting temperature at Useful Conversion Part 1 see bellow.

(1) Wallace, R.B.; Shaffer, J.; Murphy R.F.; Bonner, J.; Hirose, T.; Itakura, K.; (1979) Nucelic Acid Res. 6, 3543
(2) Breslauer, K.J.; Frank, R.; Blöcker, H.; Marky, L.A.; (1986) Proc.Natl.Acad.Sci.USA 83, 3746-3750

Click Back to Useful Conversion Part 1, click forward to go to my Molecular Biology page