5 replies to this topic

[Biocat]

Hi all,

Recently i tired to amplify one gene (low GC content: 38.1%) from genomic DNA, but didn't work, below is my detailed experiments:

Forward primer: 5’-ACTAGCTAGCATGAAAAAGAATAAAGG-3’ (add NheI restrictioin site, Tm 62; GC=33%)
Reverse primer: 5’-TTAATGATGCCATTCAACAG-3’ (Tm 58, GC=35%)
I used Roche - FastStart High Fidelity PCR System for amplification (50 ul)
PCR programme is modified with protocol from the manual:
2’-94C 1x
10s-94C 10x
70s- 50C
2.5’-72C
15s-94C 20x
30s-50C
2.5’ + 5s per cycle- 72C
7’-72C 1x
So first used 50 C as annealing temperature, didn't see the band (5 ul of PCR products with 2 ul of loading dye).
Because of my fwd primer's beginning part (restriciton site) not binding with template, i found Tm of (ATGAAAAAGAATAAAGG) just 45 C, later i used 40 C as annealing temperture, but also didn't work.
I wonder to know what's the problem in it? Could you give me some suggestions, many thanks in advance!

Biocat

[phage434]

Lower your extension temperature from 72 to 65 degrees, increase the length of your extension by 50%,  and try again.  See Su 1996, PMID 8628694.

[Biocat]

phage434, on Apr 3 2010, 05:04 PM, said:

Lower your extension temperature from 72 to 65 degrees, increase the length of your extension by 50%,  and try again.  See Su 1996, PMID 8628694.

Thank you for your suggestions.
But i tired again with 65 degree of extension temperature, and 4 min for the length of extension, but it still didn't work.
Do you think i should redesign more longer primers which make Tm higher and GC content higher?

[rkay447]

Biocat, on Apr 5 2010, 08:10 AM, said:

phage434, on Apr 3 2010, 05:04 PM, said:

Lower your extension temperature from 72 to 65 degrees, increase the length of your extension by 50%,  and try again.  See Su 1996, PMID 8628694.

Thank you for your suggestions.
But i tired again with 65 degree of extension temperature, and 4 min for the length of extension, but it still didn't work.
Do you think i should redesign more longer primers which make Tm higher and GC content higher?

You can also try different buffers.  When having trouble, I set up the PCR in the buffers I'll post below.  I can always get it to work in one of the conditions.  It's a pain to make all the buffers but once you have them you can store them at -20 and they're good to have.  The Tris buffer has different pH and all buffers should have 0.1% Triton X-100.
Buffer:      Tris-HCl(100mM) pH:       MgCl2(mM) :   KCl(mM)  
A:                   8.3:                            15:               250
B:                   8.3:                            15:               750
C:                   8.3:                            35:               250
D:                   8.3:                            35:               750
E:                   8.7:                            15:               250
F:                   8.7:                            15:               750
G:                  8.7:                             35:               250
H:                  8.7:                             35:               750
I:                   9.0:                             15:               250
J:                   9.0:                             15:               750
K:                  9.0:                             35:               250
L:                   9.0:                             35:               750

Oh, an important note, if you are doing DpnI digestion to eliminate the template, the enzyme does NOT work in the 250mM KCl buffers.  You will need to supplement the salt before digestion.  Additionally, I have seen reactions working better with the MgCl2 increased so if you get one condition to work but just not well, try that buffer (pH and KCl with 45, 55, 65 mM MgCl2).

Edited by rkay447, 05 April 2010 - 07:18 AM.

[gyma]

how many cycles did you try? and the amount of template? is your template good? does it work for other amplifications?
I think at least you can increase both the cycling number and template amount to the limit and see what might happen.

[Biocat]

gyma, on Apr 5 2010, 06:03 PM, said:

how many cycles did you try? and the amount of template? is your template good? does it work for other amplifications?
I think at least you can increase both the cycling number and template amount to the limit and see what might happen.

I used 30-35 cycles, the amount of template is 200 ng/50 ul. My template is genomic DNA, it's my first time use this genomic DNA for amplification.
I also order new primers with long linker, but didn't work as well.