I got synthesized 9 pairs of primers from GeneLink, and constituted for 100uM stock in recommended TE buffer. Then prepared working 10uM by diluting in molecular biology grade water.
Now the issue is that, I am not getting any amplification with these primers. I have tried gradient PCR by changing the annealing temperature, but in vain. The control primers are working fine with the same template and master mix.!!!
Are you giving enough time for your polymerase to cover the length of your amplicon? Perhaps different annealing times need to be considered. What is the GC content of your primers? Sometimes the secondary structure gets in the way of annealing and the use of small amounts (1-5%) of DMSO is needed to allow straightening of single strand nucleotides.
Are you giving enough time for your polymerase to cover the length of your amplicon? Perhaps different annealing times need to be considered. What is the GC content of your primers? Sometimes the secondary structure gets in the way of annealing and the use of small amounts (1-5%) of DMSO is needed to allow straightening of single strand nucleotides.
i think you should check the primers (working dilution) by gel run in 4% gel.
I would also check by using different primer concentrations and MgCl2.Have you ordered control primers at different times? There could be problem with primer synthesis.
Did you check the EDTA concentration in your TE buffer. EDTA is a chelating agent that can sequester the Mg ions needed by DNA polymerases.
Double-check your primers' sequences, to see if your designs (including their orientation) are correct.
Make sure you are giving at least 1 min for extension of 1kb PCR product. Check that you are getting same product size from control as well as test products.
We always dissolve primers in water, not in TE. I don't think TE is good, although primers are added in a low concentration. Try using a different Taq, sometimes high-fidelity Taq don't work but normal Taq works. First optimize the condition with a normal taq.
What are your control primers? First of all, you may want to make sure if your primers are correct? Then second strp would be to optimize the MgCL2, dNTPs and Taq polymerase concentrations which are usually recommended by the PCR kit you would be using, but the use of 7.5% DMSO would help specific annealing. The only thing remains is the extension time. It is recommended that 1 kb/minute is required for complete extension.
By using the right concentrations of buffers and enzyme and fixing your extension time, your PCR reaction should work. And if your DNA template is good (I mean if don't have inhibitors) then you should be fine. If still it doesn't work then believe me your primers are wrong. You need to redesign them. Pay attention to the reference genome you used to design the primers. Different strains of the same organism would affect the amplifications.
Hii..
Are you sure that, you are using the PCR ingradients with final concentration:
1X PCR buffrer, 0.2-0.5 mM dNTP's, 0.2-0.5uM primers, 1.5-2.0 mM MgCl2, Polymerase 1-2 units (Some PCR may works well from 0.2 units)..
If it is fine then play around with different DNA concentrations (10ng-1ug) (for universal primers, 10ng DNA is enough but for degenerate primers/single copy genes you may increase DNA concentration).
Also most of the PCR do not work well because of impure DNA samples compared to control. In this case purify sample using column or you may use enhancers like 0.2-0.5% BSA or DMSO...
Hope these thing may solve your problem..
Best wishes..
Make sure of following things:
1) anneling temperature is correct
2) time for product elonganion is enough (polymerase usually build chain near 1000b.p. per minute)
3) MgCl2 concentration is enough
4) the direction of syntysised primers are right
5) DNA for PCR is ok
6) all reagents of PCR-mix is ok
In case if all of these things are ok, try touchdown PCR. It is very good and helpful thing. Also you may use DMSO.
I agree with Atiyeh, the primers are not annealing with the template.
Check the sense or polarity of the primers. If they are convergent or wrongly divergent...
If every thing is good with your positive control ....... Means just primer issue, then if primer sequence is ok. Y not to check from the company you synthesized them.
Yes, sometimes companies who synthesize primers may do mistakes. I don't know how reliable your source is. Double check everything. Good luck,
RC
Please find attached files;
https://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf
http://genepool.bio.ed.ac.uk/sanger/Sanger_troubleshooting_guide_v1.pdf
http://palumbi.stanford.edu/SimpleFoolsMaster.pdf
https://www.biocompare.com/2-DNA-Forums/1315-Problems-with-cDNA-gene-amplification/
https://www.operon.com/services/dna-sequencing/troubleshooting.pdf
I hope these files will help you to solve your issue.
Regards
Ali
@ Muhammad Shakeel I had the same problem and drew new primers. Problem solved!
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