I have a problem with amplification of a specific gene. After gene specific primer designing, I amplify my desired gene with PCR but don't get any answer. My enzyme is DreamTaq DNA polymerase. I tested a different protocol and I tested my kit too but none worked. So do you think my primer is bad? How can I solve this problem?
Dear Mahboubeh Mostafaei, The problem is with your primer. As a first step, Check the specificity of your primers using softwares like Primer Blast or Serial Cloner if tou have your target sequence
Hi MM, PCR results not only depends on the Taq and primers you are using, it also involves others important things like concentrationof pcr components, the conditions you are following and so on. At each and every step you need to be very careful start from primer designing till visualising gel under uv. My suggestion is, you just keep on trying different combination of the required ones to get expected such as design different primers, use different combinations of concentrations and conditions, hot start, touch down pcr, gradient pcr etc. Good luck.
As suggested above I would suggest :
1. Check your M.M water for any contamination.
2. Check your diluted primers and template on gel .
3. sometimes thermal cycler also creates some problems , instead of going for single 50 microliter reaction , make two tubes of 25 microliter.
4. run controls both negative and positive.
hopefully this would work
All the Best
Hi Mahboubeh, as quite clearly proposed by colleagues, primers are indeed one of the critical components, design first and concentration secondly, so I will not say much more.
But check also your template!
If you are using genomic DNA, be careful not to concentrate it too much (do not use more than 300 ng) otherwise you inhibit polymerase function, and try primer concentration up to 1 uM.
If your template is cDNA, you should be sure it was produced from good quality RNA, otherwise you will probably have too few copies of your full length gene. You can try ctrl PCRs as suggested, but if your gene is low expressed compared to GAPDH or other housekeeping genes you might not solve the problem...
Hi, all the above suggestions are fine but please do tell me your DNA size and PCR conditions as these may be a great deciding factor for PCR. Did you try your PCR components for any other housekeeping genes and did it work?
Thanks all of you.
My product size is 1638 bp and about PCR condition: 3 min at 95oC- 35 cycles of 30 second at 95oC, 30 second at 55.5 oC and 2 min at 72oC. 10 min at 72 oC after 35 cycles. Before this I got answer with this annealing temprature, my primer finished and I had to use from primer stock solution and dilute it again but it didnt work.
You know I didnt get answer for housekeeping but I tried it with kit and I got answer. I tried for my desired gene with kit too but it didnt work.
First check the quality of your DNA. If it is fine then PCR should work. Then primers should be fine. Some times old primers may degenerate, (frequent thawing). And the ingredients should also be in tact.
Could you please upload your DNA gel picture.
Dear,
1) used this profile for mentioned gene size : min at 95oC- 35 cycles of 30 second at 95oC, 30 second at 55.5 oC and 3 min at 72oC. 10 min at 72 oC after 35 cycles.
2) do this by gradient PCR
3) and use manual Taq for PCR reaction
4) use primer sequence by primer3 to check the compatibility of primer then go for PCR
Hi Mahboubeh!
First of all you have to be sure of the company which synthesized your primers. Maybe the Primer synthesis was not satisfied.
As you know the concentration of Mg ion is critical in PCR. you can change your Mg concentration or add a little bit of it.
10 pM concentration of primers would be appreciate.
Gradient PCR would be beneficial to achieve your goal.
I suggest if you could not get the expected results go for another primer design with primer3 software and synthesis it by other company rather the previous one.
Good luck!
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