6 replies to this topic

[drollix]

Hello,

I have been trying to amplify a region from genomic DNA using 60 bp primers (Tm = 65 deg C), and it's just not working i.e. no products. The primer sequences are from a papers, and they check out. The entire primer sequence is homologous to the template. I am using Amplitaq gold pre-mix with annealing temp of 60 degrees in a regular 30 cycle PCR mode.
The options for troubleshooting that I have are:
1) Use another enzyme or mix. Amplitaq gives a product on positive controls.
2) Use another PCR protocol (touchdown PCR)

The reason I am using long primers is that 5' region of the primer length should match a genomic region in target strain for homologous recombination.

Has anyone been through this problem?

[pcrman]

What is your PCR product size and the size of another primer? If the whole primer is homologous to the template (no mismatches), then what is the point of using such long primer?

One thing you may want to check is the primer quality. You can simply run a agarose gel to see if the primer gives you a band at 60 bp.

[drollix]

pcrman, on Apr 9 2009, 11:57 PM, said:

What is your PCR product size and the size of another primer? If the whole primer is homologous to the template (no mismatches), then what is the point of using such long primer?

One thing you may want to check is the primer quality. You can simply run a agarose gel to see if the primer gives you a band at 60 bp.

The PCR product size is 1.5 KB. The primer is long, even though its perfectly identical to the template sequence, because the product is designed as having 3 contiguous regions A-B-A, and only the A region (45 bp at both ends of the product) will be homologous to the target genomic DNA sequence.

[phage434]

I'm sorry, this makes very little sense.  Could you please list:

(1) the primers you are using
(2) the sequence, portion of sequence, or ID of the sequence you hope to amplify
(3) your expected product sequence

[lnorwood]

Have you tried adding DMSO to your PCR reaction?  With large primers you can get primer secondary structure that is relieved a lot by DMSO addition.  Also, you could try a touchdown PCR (annealing temp changes 0.5-1 degree each cycle).

drollix, on Apr 9 2009, 02:24 PM, said:

Hello,

I have been trying to amplify a region from genomic DNA using 60 bp primers (Tm = 65 deg C), and it's just not working i.e. no products. The primer sequences are from a papers, and they check out. The entire primer sequence is homologous to the template. I am using Amplitaq gold pre-mix with annealing temp of 60 degrees in a regular 30 cycle PCR mode.
The options for troubleshooting that I have are:
1) Use another enzyme or mix. Amplitaq gives a product on positive controls.
2) Use another PCR protocol (touchdown PCR)

The reason I am using long primers is that 5' region of the primer length should match a genomic region in target strain for homologous recombination.

Has anyone been through this problem?

[gfischer]

1.5 kb seems like an awfully long product.  Would it be possible to redesign you primers for a shorter product?  If not, make sure the extension step of your PCR is long enough.

[swanny]

drollix, on Apr 10 2009, 07:24 AM, said:

Hello,

I have been trying to amplify a region from genomic DNA using 60 bp primers (Tm = 65 deg C), and it's just not working i.e. no products. The primer sequences are from a papers, and they check out. The entire primer sequence is homologous to the template. I am using Amplitaq gold pre-mix with annealing temp of 60 degrees in a regular 30 cycle PCR mode.
The options for troubleshooting that I have are:
1) Use another enzyme or mix. Amplitaq gives a product on positive controls.
2) Use another PCR protocol (touchdown PCR)

The reason I am using long primers is that 5' region of the primer length should match a genomic region in target strain for homologous recombination.

Has anyone been through this problem?

We really need more details. What temperatures are you using?

With no other information, my guess would be your annealing temp range is too high. Why do you think the Tm is 65? Try the old 2(A+T) + 4(G+C) approximation. I'd try 50 - 55 degrees.