no amplification

 I am trying to amplify a gene of 1359 bp. the primers that i have designed are AT rich. The forward primer has a strech of A and T which results in formation of hairpin and dimers. I am not getting any amplification. what should I do??

Hi monisha,

There are a number of things you can do. Here are some suggestions: 

1. Increase the extension step of your PCR reaction. 
2. Design new primers; the primers you have may not be good for PCR amplification, as you eluded to with the AT-rich, hairpin and dimer comment. 
3. Obtain a new batch of DNA template. Your DNA may not be of good quality, or of high enough concentration.
4. Try to amplify a smaller portion of the gene first to see if it can be amplified. Amplifying a ~500 bp fragment is a lot easier than amplifying a 1,359 fragment. Instead of struggling to amplify a large fragment, first make sure the gene you are trying to amplify does not have some weird DNA that makes it hard to amplify. 

Good luck

 thanks Ivan...
have a doubt...what can make a DNA  fragment hard to amplify....i mean what does "weird" in ur answer refer 2???

Just like primers can have hairpins and dimers, DNA can form all sorts of three dimensional structures that can make PCR hard if not impossible. For example a stretch of GC-rich DNA is known for being hard to amplify, which is why there are PCR reagents out there specifically designed to amplify GC-rich DNA.

 okk....got ur point....but my template is not that GC rich....i guess i shud change my primers......

monisha,

The GC-rich example I gave you was just an example. There are many other reasons why your DNA may not be easy to amplify, like methylation. In addition, having DNA that is not easy to amplify is also just one of the many reasons why your PCR is not working. As I mentioned in an earlier email there are many other things you may want to consider, like making sure that your primers are working by designing new primers, or primers that amplify a smaller DNA fragment. 

 thanks Ivan
i have designd new primers ....i hope they work....

Hi Ivan,

I was reading your conversation with Monisha regarding PCR amplification. While reading i got  a doubt, i hope you would not mind to answer it. Her gene size is 1359 bp, then how can she start with small gene, i mean as you mentioned amplifying a fragment of 500 bp is easier yaa that is fine but her gene is not as small. She has to amplify the whole gene(~1359bp). Please make it clear to me.


Thanks
Regards
Rupsi Garg

Hi Everyone,

It really depends on the purpose of amplifying the entire gene. If it is simply to perform Sanger sequencing to find the exact sequence, then smaller PCR is a plausible direction. If the entire gene is required then it is better to determine the cause of no amplification. But, as Ivan stated, breaking down the PCR into smaller fragments could help determine if the problem is related to the actual sequence and not PCR conditions. Unfortunately, amplifying smaller fragment would require some knowledge of the actual sequence of the gene.

Hairpins are exceptionally problematic. We have seen hairpins in sequencing that hide 2 kb in one direction, but not the reverse. There are certain PCR additives that could help if there is a hairpin. Betaine sometimes works.

Monisha, could you provide the sequence of the primers? Maybe we see the problem in the sequences.

DougB

Hi Rupsi,

I believe DougB answered your question. If you have a gene that is 1,359 bp, there is lots of space for DNA sequences that could make it hard, if not impossible, for a given Taq to amplify. By PCR amplifying smaller regions you increase the chance that you may amplify a region that is much easier to PCR amplify.

Of course if the whole purpose of the experiment is to amplify the whole gene, then you amplify the gene in two or three pieces and then clone them together. This would be hard too, but if amplifying the whole gene in a single PCR is impossible, then amplifying the gene in smaller pieces may be the only way to go. This has been done in the past for genes that are simply too hard to amplify in a single PCR. 

I hope this clarifies things.