8 replies to this topic

[light_my_way]

Hi all,

I am an amateur in doing PCR. Recently i did PCR for a genomic DNA, hoping to get a 322bp DNA sequence. After I carried out a gel run, I realised my desired band was faint and there were many prominent primer-dimers. I thought it was because the template concentration was low. So I PCR-Clean using Qiagen and used the eluent as template for another round of PCR. However, the primer-dimers were still present. May I know what could be the cause?

[nabla]

Hello! Have you had a look at this thread?
Greetz nabla


http://www.protocol-online.org/forums/inde...?showtopic=4099

[light_my_way]

Hi. Thanks nabla for the link. But I saw this post by vgupta. He/She says that we should try Mg2+ at a final concentration of 1.5, 2.0, 2.5 and 3.0 mM. However, how should I carry out the titration?

By the way, I have already done a temperature gradient. But there are still primer-dimer present for each temperature.

Edited by light_my_way, 13 February 2005 - 06:58 AM.

[tfitzwater]

Please supply the sequences of your primers.

[light_my_way]

Hi,

My two primers are:
1) 5'-cgggatccatggatggtatggat-3'
2) 5'- ccgaattcgctggaagaactaca-3

Is the primer-dimer problem due to my designed primers?

[ger225]

Yes, you have a major primer-dimer problem. Your first primer sequence is bad, the primer forms not only primer-dimers but also hairpins. The problem seems to be the last 5 nucleotides (´3). Change them!!. The other primer seems OK!

ger225

[light_my_way]

Hi,

Thanks for the advice. However, how do I know if the next primer I deisgn is free from hairpins problem? Is there a way to do it?

[ger225]

Hi!

Well, there´s not an easy way to check your primers only by yourself. However, you must always check if the 3´end anneal with any other sequence located in both primers, because the polymerase you use will start adding nucleotides there, forming primer-dimers. For example, in your primer Nº 1, the sequence 5-ATGGAT-3 anneals perfectly with 3-TACCTA-5 located in the same primer. That leads to the formation of primer-dimers and if the primer folds backs on itself, it will form an stable hairpin.
To check your primers easily, the best way is to get help from a computer program. We use Dnastar, which allows you to enter the sequence of your primers and informs you whether they form primer self dimers, primer pair dimers and primer hairpins. I don´t know if you can get it in the Internet but I´m almost sure that you can find free software in the WWW to check your primers. If not, talk to someone in the Molecular Biology Deparment, these programs are used all the time in these labs!!
Good luck!

[sharath]

If primer dimers is such a meance, you can go for gel purifation of your smaple. The DNA is run in a low melting agarose gel and extrated wih glass beads. There are standard kits for this.