I used 8 primers and two samples but no DNA band obtained.
I also vary the temperature gradient fro 46-54 but still not getting any bands.
are you sure that your samples can amplify anything ( dna ok) and have your target sequence?
Are you seeing size standard or any signal at all on your gel.If so can we see a picture please? Some information about pcr conditions and ideally a primer pair sequence and what the target dna source is would be helpful
I'd choose one pair of primers to start with and once you get those working, then test the others. It's hard to know what the issue is without any additional information. Do you have a good positive control sample to use? Do you know your expected product size for each pair of primers? If anyone else in your lab uses these primers, ask them for their thermocycler conditions.
Paul is right. We need a good deal more information before we can offer any constructive suggestions.
Possible causes of failure for a PCR reaction are:
(1) Faulty instrument
(2) Inactive mastermix/polymerase
(3) Defective primers (this tends to happen batchwise when manufacturers are having problems with their synthesisers
(4) Samples inhibitory, degraded, or no DNA present
(5) Failure of detection
You need a control PCR which you know will work and a control sample that you know is amplifiable in order to eliminate (1), (2) and (5).
Then test your samples with the control PCR. If you don't get a band, try adding control DNA to the sample. If you still don't get a band, the sample is inhibitory.
Andrew
This result I suggest due to the quantity of DNA is few in solution ..so I suggest for good result incraese the concentration in solution
If you are not getting results after several attempts.
1. Check DNA on 1% Agarose Gel or Spectrophotometr for quantity of DNA.
2. Check your primers (Stock+Dilution).
3. Go for optimization of one marker then go for others.
you can use other method of DNA extraction ,and check for the quality of the DNA through Farouq's suggestion.
moreso,you can try out few Random polymorphic markers.
Increase the amount of DNA and primers u will get an amazing results. And make sure firstly the reconstitution of primers is correct.
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