HI! whenever I start doing PCR with a new primer pair that is lyophilized.1st I make 100pmol solution then 10pmole dilution of it as a working solution. I got good results with this freshly made 10pmole dilution but only one time if I use it just after making dilution of lyophilized primers. after 1or 2 days these primers do not work at all. I have autoclaved each and every plasticware like tips, PCR tubes, Eppendorf tubes, again and again, to resolve all sterility issues but again no results. After my early experiments, I thought I should use nuclease-free water instead of injection water ampules but all in vain, I have got the same problem. what could be the possible reasons for not having PCR results. what I should do in this scenario? suggestions, please
Some people suggested that don't use water at all. instead of that, you can use TE buffer. The Tris and EDTA prevent acidic water and contaminating DNases from hydrolyzing and enzymatically degrading DNA, respectively. and where you keep the primers?
Did you check the primers on agarose gel? primer degradation leads to smear in your gel.
yes, I have checked my primers on 2% agarose gel. gel image is attached here please find. In the first four wells I have loaded 10mol dilution and in next four wells 100pmol dilution of these primers. please analyze.
I have some suggestion for you to be sure that the problem is not related to your primers.
1. keep your primers (stock and working solution) at -20.
2. Use RNase-DNase free mictotube
3. make two group and for first one add TE buffer as a solvent and add water ( nuclease-free water or injection water ampules) for second one then do PCR directly. If you get result, keep the primers at -20 then try again after few days.
Are you using the same genomic DNA? if no, please check the concentration of genomic DNA, being used in PCR reaction.
will you please upload agarose gel, run with the PCR products the second time, where PCR has failed. Looking at the gel, some inference can be drawn.
Always keep your primers both working and stock at -20°C. Even when preparing PCR reaction mix, keep your primers in chilled condition (not above 4°C) using mini cooler or ice.
Dear Nargis, please change loading dye, as it is contaminated with gDNA.
Primers seem ok.
I think pcr product is less than 1 kb, if primers are 5 mol, increase the gDNA concentration, like 1ul, 2ul and so on to see which concentration is working fine. Generally in a 25 ul reaction, 50 -100 ng template DNA with 5/10 pmol primer work fine.
Hello Nargis Afzal , start fresh by making working stocks of primers in nuclease free water. How did you extract your DNA/RNA? Is it free from PCR Inhibitors. If you think your extracted sample may have PCR inhibitors then try diluting your sample with nuclease free water and then perform PCR. Try increasing your PCR cycles to 45, you may be having lower amount of template. Try adding more template to your PCR reaction, 5 microliters of your sample don't add more amount than this in your PCR reaction as it may cause PCR inhibition. Also try loading more sample 15 microliters if possible in our gel. If these steps don't work try using a different PCR master mix.
I, hope these suggestions will let you see your desired PCR bands.
I wonder whether you are irradiating your primers with UV light during set up ...
I cannot see unused primers in your gel, is it true? That means that they have been used. Try to amplify a second time your PCR using 2ul of the sample, may be your templete has inhibitors. If this does not work, try to use a little more template. good luck
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