How good are my PCR primers? can I go with it?! - (Dec/06/2007 )

Hi folks,

I am new to the area of PCR cloning and am trying to amplify a 3000bp region from a plasmid construct. I use Invitrogen VectorNTI's (amplify selection) method to design a primer. What i get now is primers of 20bp length (both sense and antisense)with these calculated values
Contains region of the molecule from 13 to 3001
Tm: 80.7 C TaOpt: 55.3 C GC: 51.3
Sense Primer:
TAACTGGTTTCCTGAAAGGT
Similarity: 100.0%
Length: 20 Tm: 45.6 C GC: 40.0
dH: -150.2 kcal/mol dS: -394.6 cal/mol dG: -30.8 kcal/mol
Antisense Primer:
CCCATTCCAGCGCCAGGAGC
Similarity: 100.0%
Length: 20 Tm: 63.4 C GC: 70.0
dH: -169.9 kcal/mol dS: -427.7 cal/mol dG: -40.6 kcal/mol
Tm Difference: 17.8
GC Difference: 30.0

Is this alright to go with? or should i play around these values. Suggestions are very much appreciated. Thanks in advance

you may want to try to match tm a little better and maybe reduce the g-c content of the antisense. you may also want to end your sense primer with a g or c.

you don't need to restrict the size to 20 bases.

i agree with mdfenko.

your antisence should have a lower Tm.
also i tested your primers in my program and thats my results for primer1, primer2 and the combination of 1 and 2.
I think you can try and make better...


Name : Primer 1
Primer : 5΄-TAACTGGTTTCCTGAAAGGT-3΄
Reverse : 3΄-TGGAAAGTCCTTTGGTCAAT-5΄
Length : 20 nt

Tm (basic) : 56,0 ΊC
Tm (salt) : 54,3 ΊC
Tm (NN) : 55,5 ΊC

GC % : 40,0 %
dG : -36,5 kCal/mol

3'-tail GC % : 42,9 %
3'-tail dG : -9,4 kCal/mol

Molecular weight : 6209,0 g/mol

1 ml of the primer solution with an
absorbance of 1 at 260 nm is 4,63 µM
and contains 28,7 µg ssDNA


Primer 1 self annealing:

5'-TAACTGGTTTCCTGAAAGGT-3'
||| :::
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -2,01 kcal/mol

5'-TAACTGGTTTCCTGAAAGGT-3'
|||| ::::
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -1,74 kcal/mol

5'-TAACTGGTTTCCTGAAAGGT-3'
||| :::
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -0,29 kcal/mol


Primer 1 loops:

5'-TAACTGGTTTCCTG
||| A
3'-TGGAA
dG: -1,59 kcal/mol

5'-TAACTGGTTTCC
||| )
3'-TGGAAAGT
dG: 1,40 kcal/mol




Name : Primer 2
Primer : 5΄-CCCATTCCAGCGCCAGGAGC-3΄
Reverse : 3΄-CGAGGACCGCGACCTTACCC-5΄
Length : 20 nt

Tm (basic) : 68,0 ΊC
Tm (salt) : 66,6 ΊC
Tm (NN) : 69,6 ΊC

GC % : 70,0 %
dG : -47,1 kCal/mol

3'-tail GC % : 71,4 %
3'-tail dG : -12,6 kCal/mol

Molecular weight : 6109,9 g/mol

1 ml of the primer solution with an
absorbance of 1 at 260 nm is 4,97 µM
and contains 30,4 µg ssDNA


Primer 2 self annealing:

5'-CCCATTCCAGCGCCAGGAGC-3'
: : |||| : :
3'-CGAGGACCGCGACCTTACCC-5'
dG: -6,66 kcal/mol

5'-CCCATTCCAGCGCCAGGAGC-3'
||| : : :::
3'-CGAGGACCGCGACCTTACCC-5'
dG: -2,85 kcal/mol


Primer 2 loops:

5'-CCCATTCCAGC
||| : G
3'-CGAGGACC
dG: -2,43 kcal/mol




Primer 2 - Primer 1 annealing:

5'-CCCATTCCAGCGCCAGGAGC-3'
|||||
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -6,95 kcal/mol

5'-CCCATTCCAGCGCCAGGAGC-3'
:: : ||||
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -4,22 kcal/mol

5'-CCCATTCCAGCGCCAGGAGC-3'
: ||||
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -4,22 kcal/mol

5'-CCCATTCCAGCGCCAGGAGC-3'
||| :
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -2,35 kcal/mol

5'-CCCATTCCAGCGCCAGGAGC-3'
: :: |||
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -0,88 kcal/mol

5'-CCCATTCCAGCGCCAGGAGC-3'
:: ||| : : :
3'-TGGAAAGTCCTTTGGTCAAT-5'
dG: -0,62 kcal/mol