Dear Shahed,

If your PCR works fine otherwise, then it seems your target DNA concentration was too low with your recent samples. Try to extract again or if you still have the extracted DNA, try to measure DNA cc with Qubit or Bioanalyzer.

Good luck!

Yeah...the problem might be linked with DNA concentration variation. Try to do PCR by varying the sample concentration. It may be too low or too high from the previous one.Higher conc DNA can hinder your PCR also. You can also check it with nanodrop along with the above suggested instrument. Best of luck !!

I usually check with a nanodrop (like Debaprasad Parai said) and use all my DNA at 40ng/ul. Try it at that concentration or all at the same consentration.

I will give you two suggestion :

1-you can try by increasing both the DNA sample concentration and primer concentration.its helpful for you.

2-your primer dimer intensity to high so you can increase BSA concentration if you are use BSA in in your PCR reaction.

Best of luck.

You need to check:

Concentration and Purity of your DNA (40-50ng for your PCR, A260/280 = ~1.8).

You can increase Mg2+ in your buffer

All the best

Sounds like the perfect time to do a mixing experiment (which will tell you if there is something inhibitory in your DNA preparation that may by effecting the PCR reaction)

Set up 4 reactions:

1) PCR containing all of the reaction components but lacking the DNA template

2) PCR of your target region with a DNA prep you know works

3) PCR of your target region with the DNA prep in question

4) PCR of your target region with the DNA prep that works + the DNA in question

If reaction 2 works, but 4 doesn't - that means you have something inhibitory in your DNA prep. If that is the case I would recommend: re-extracting with phenol-chloroform, then chloroform, then precipiate and wash the pellet with 70% ethanol.

That should do the trick

The gel picture tells that the template DNA is well in the range that can give you some positive result if the rest of the procedure is fine. If you still doubt about the DNA concentration, just pool a few samples and use them for PCR. Use some stabilizer in PCR just like BSA etc. Choose the proper enzyme and conditions according to the length of the target you want to amplify

I have changed the polymerase enzyme and now it is working ,,

hi dear

u must test the quality of yours DNA by agarose gel electrophoresis and also measure the concentration of your DNA .

if yours DNA is highly smeared you must extract anew DNA because the smeared DNA indicated that your DNA is degraded and its not suitable for PCR .

also if the concentration of your DNA too hi you must dilute it but if too low repeat the extraction

finally if the concentration of the DNA acceptable for PCR check the present of inhibitor in yours DNA by diluting it 1:10 with increasing the volume of DNA in yours reaction and decrease the concentration of yours primers in the PCR reaction .

CONTENUE

if the problem not resolved check the quality of your primers if too old or stored at -18 for long time make heat shook for both primers and also avoid contamination in yours lab

best regard