help needed: PCR a gene from genomic DNA - (Jan/25/2010 )

Hi everybody,
I am trying to amplify a gene from a plant species. This gene has two copies, one has a intron, and the other one dose not. I wanted to amplify whole gene of both copies from the genome, however, only the none-intron copy can be amplified.
Here is the detail information of the experiment

the none intron copy is about 1.3 kb, and with intron copy is about 2.5 kb

DNA was extracted by Qiagen DNA extraction kit, and quality of DNA has been checked on Nanodrop.

Advantage GC 2 DNA polymerase from clonetech was used to amplify this gene from genome.

Touch down program was used for PCR, 96deg 15sec, 3X(94deg 25sec, 68deg 3.5min) 7X(94deg 25sec, 67->60deg 30sec, 68deg 3min) 30X(94deg 25sec, 58deg 30sec, 68deg, 3min)


I can amplify each exon independently, and intron as well by using primers on the edge of exons. However, when i used designed primer from end of the gene, only the non-intron copy can be amplified.

I've check the intron sequence i got, it was quit AT rich, (~63%AT, 37%GC)

Anybody has clue how to solve the problem?

Thanks a lot.


St.

ophrys on Jan 25 2010, 08:48 PM said:

Hi there,

It is possible that your PCR product containing the intron is a wee bit too large, so it is not amplifying well... I take it that you're using the same primers to attempt to amplify both products and would like to see 2 products when you run on a gel - one at 1.5kb and one at 2.5kb?

When trying to amplify a larger product, you do need more reagents (eg: dNTPs) which are normally in excess when amplifying a small (say 500bp) product, but might be limiting the reaction for a longer prodcut... The Taq that you are using might also not be suitable for amplification of such a large fragment (they can get 'exhausted' (!!) from amplifying such a large fragment) so maybe check with the company whether it's suitable - you might need to switch to one more suited.

There are definitely products out there that will work better for longer fragments.

Finally, you could also thinking about extending your annealing and extension times ... they're long fragments, so need longer to create! Maybe use a program more like: 95oC - 30sec, 67 --> 58oC (touchdown) - 1min, 72oC - 3min

This post might also have some more ideas for you to try http://www.protocol-online.org/biology-for...posts/4384.html

Good luck!

The first place I would start is adjusting your extension time (although 3min should be plenty for taq for 2.5kb)

I don't ever add more dNTPs to my longer product reactions and this has never been a problem (even for my 4kb products) so I doubt that is it.

Could you try a gradient rather than a touchdown?

Also, your annealing temps seem rather high to me- what is the Tm of your primers. Perhaps you could try a gradient from around 55C right up to around 65 and see if you have any luck.

Good luck