I am not getting amplification of my gene. Its gc content is 53 % do I need to use supplementary substances like DMSO etc, its Tm is ~62 degree. the primers have been designed with restriction sites at the 3'end, will that influence the melting temperature. Upon adding these sites the GC content becomes 55 and 61 %, and the Tm becomes 71 and 78 degrees for forward and reverse primers respectively. I do not know which Tm to use for setting up my reaction. I am using Clone manager for primer designing and carrying out gradient PCR.
1) Double- or triple-check the sequence you want to amplify (different sources exist beyon only NCBI :) ).
2) The most reliable Tm calculator, IMHO, is this one: http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ . It allows for very precise determination of Tm, depending on the conditions of your PCR.
3) Use gradient PCR first, then in second run MgCl2 gradient in the best Tm, then in the 3rd run primer gradient in the previously optimized conditions.
Hi Sheetal,
The link given by Marcin is really good for checking the thermodynamics of your primer.In this you can check for the Tm, GC content, hairpin loops, self dimers, heterodimers etc..
Try to check, if the primers are fine (assuming that the target sequence is correct).
Bye the way, what,s the annealing temperature you are using for setting up the reaction???
Regards
Sushma
I have had such problem before. You might need to cross check the target sequences that you want to amplify. Again you can download serial cloner and use it to check whether or not you will have a correct PCR product before you repeat the experiment.. The template DNA might also have been contaminated.
Hi.
While calculating the Tm you need not include the added restriction sites. So just re-check your primer sequence and try doing pcr optimization.
Good Luck!
As always, it would help if you provided relevant information. What is your target? What are your primers? Are you detecting, quantifying, cloning?
Thanks all. The size of my gene is 3KB, the anealing temperatures that I have used are 55+- 5 degrees gradient, another one at 60+- 5 degrees. I want to clone this gene. I e have designed primers using the CDS of this gene, using the first and last 20 bases. to get the complete gene, if I were to redesign my primers do i still have any choice of which bases to take as i need to amplify the complete gene which i want to clone and express in another organism.
Which concentrations of MgCL2 , dNTPs, primers would be optimum to start with using a gradient PCR. Presently I am taking 2mM MgCl2, 0.2mM each dNTP, 1pM primers, about 100 ng DNA.
Sheetal,
If direct CDS cloning fails, you may do half-and-half: Look for a RARE (single cut within CDS) restriction site, somewhere around middle of the sequence, and design primers for cloning both halves, in individual separate reactions. After cloning 1st half in a plasmid, you amplify second half, cut out the excess from a subclone (SEQUENCE IT before!!!) (double-digest!), and ligate in its stead.
2mM MgCl2 per reaction is a good starting point, although it may vary from 0 to 5mM, so be warned.
Primers: Usually 0.1-0.6uM; I start out with 200-300nM.
dNTP: usually 50-500uM, normally Id start with ~200uM to have margin on either side.
100ng DNA suggest you clone it from gDNA; I think you can go as low as ~30ng/reaction.
Hope this helps,
Marcin
Thanks Marcin.
When I went for primer conccentration of 10 picomoles I got primer dimers. mgcl2 ws 2mM, dNTP 0.2mM,
Thanks a lot Arthur, i am now redesigning primers so that the gene will be isolated in two smaller fragments and later ligated to get the fully functional gene.
Check your primer.. During design you need to avoid maximum chances of primer dimer formation, false priming and loop formation because your gene has high G-C content.. Try some other software for primer design and do a blast with NCBI Primer Blast, then you proceed...
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