You have to increase primers melting temperature in your PCR. It's good to use temperature gradient if you have this option in your thermocycler
Hi, yes, try a gradient PCR (a range of different annealing temperatures), try some more cycles, try a final elongation step for 5 min and try to optimize our MgCl2-concentration. All the best, Nadine
Gradually increase the annealing temperatrue of primer. If it not worjs, decrease the concentration of Mg.
Hi, try also to optimize primer concentration. Primers in excess are annealing one to each other, or annealing to non specifique DNA.
Other than increasing the annealing temperature and/or decreasing MgCl2 concentration, all excellent suggestions, you could try diluting your DNA (e.g. 1:10) to dilute access background DNA. That helps in particular situations, such as trying to amplify bacterial DNA in a background of eukaryotic DNA.
Keep us posted to see if you got any improvement!
Make your reaction mixture on ice. Keep on ice until you are ready to load the tubes into the thermocycler. If at all possible, transfer your tunes to a hot thermocycler (95 C). This will substantially reduce the generation of products that result from short complements of subject DNA with the primer 3' ends.
If you know the sequence you are amplifying, you can add a 4-cutter restriction enzyme to destroy many of the sequences that might cause a problem. (If this works, remember me with kindness).
Too much DNA can result in split peaks or peaks that are off scale.Too little DNA template may result in allele drop-out.Dilute ur DNA sample.STR kits specify the addition of between 1to2.5 ng of template DNA for optimum results.
Two important tips, first of all, read more about PCR, what conditions do you need, also, what additives you can use. Second one, you have to use a good Taq enzyme, like Roche, that I use. Every technique that you'll start to use in molecular biology, you have to know everything about it! Because, if not, you only try cooking recipes. Good luck!
Did you check insilico PCR with your primer? Because non specific primers might leads to non-specific bands. How you designed primer? Did you use any online primer designing tool or manually you designed primer. Because primer designing might also be responsible for non-specific bands.
Firstly, try and check if your primer length is optimal. Increasing the length of the primer may make the reaction more specific, as shorter primers are more prone to non-specific binding.
Also try and do a gradient PCR to see if in creasing the annealing temperature will help get rid of the non-specific bands. Decreasing the Mg2+ concentration may also help in this regards.
If nothing works, you may have to go back to the drawing board and try designing a new set of primers.
You could try to reduce the amount of template and also do a blast on your sequence to see if the primers are annealing with other parts.
As already indicated by Jon Ashley and in addition to all the suggestions already made check you primer concentration. The standard supplier provide primers at 100 pmole/µl and it seems to be a standard lab routine to use 0.5 or even 1 µl for a 50 µl PCR, bringing your final concentration up to 1 or 2 µM. I had a number of PCRs recently where all unspecific bands disappeared without a trace after adjusting the primer concentration to 1/10 (0.1 µM), as recommended by NEB for standard Taq polymerase.
You have about every suggestion you might need to get good and specific amplification. So, I recommend you spend enough time to read through PCR protocols and trouble shooting; check the primers you are using using online programs to ensure they have no problems; blast the primers with the sequence of the template you are using to ensure the primers dont anneal to other regions; if they do, increase the primer length to make them more specific or design the primers at a different region if it does not affect the outcomes you expect; make sure you take into account the Tm of the primers to determine the temperatures you will use in the PCR reaction and try raising and decreasing gradually depending on the starting Tm and the length of the expected product. Important, use as little DNA template as possible. About 0.5-1ng of template should work. I am sure you will have specific amplification at the end and hopefully, this can be your best opportunity to get your next PCR successful at a go. Molecular work demands a lot of patience and skillful thinking. Welcome and Good luck!
I have tried using touchdown PCR where you can use annealing T of 10 C higher than the lowest Tm of your primer after the initial denaturation cycle, decreasing the annealing T by 1.3 C every 2 cycles and extension at 68 C, for a total of 18 cycles. Then anneal at the lowest Tm of your primer, 68 C extension for about 25 cycles. Secondly try adjusting the MgCl concentration.
First of all do an ePCR to check primer specificity.
If it gives specific product that you are looking for, try DMSO in your PCR mix.
increase the annealing temperature, try a few degrees each time you try. Change your primers so that they are more specific.
Repeating always the same answers isn't very useful. And: check the date. The question was asked 6 month ago! Cheers, Nadine
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