Does the reamplification of the PCR product need any alterations in the original PCR components and procedure to get better and reliable amplicons? Also how best the issue of insufficient quantity of PCR product can be solved by reamplification?
Re amplification must be treated with care. In early stage pcr every 10 cycles is 1000 times more product so if you use too much template or too many cycles the amplimer will self anneal and form smears of lonmers. I would use 1ul of first round product...dilute it into 100ul and use 1ul of this dilution. Set up 6 tubes of pcr and set for 24 cycles and remove a tube at 12,14,16.....24 cycles and see which cycle number gives good clean amplification.
A cleaner way is to design an internal primer set just inside the first pcr primers. They can be quite poor as primers because the template is almost all the correct amplimer.. Run 20 cycles of a 1% dilution of the first round product ( 1ul of 1/100 dilution) and it should give a clean product. There is no need to clean up the first round product between amplifications
Agree with paul Rutland suggestion including use high fertility taq DNA polymerase it helps to prevent altration during pcr
You can get clean bands, but as Paul Rutland said, you have to work properly preferably in another room to avoid cross-contaminations and reduce the concentration of DNA template by making dilutions.
You can make dilutions of original PCR products such as 1 ul of Neat, 1/10 and 1/100 dilutions and use for reamplification, run on gel and see which gives you best amplification.
After reamplification, clean up PCR and elute in lower volume of buffer e. g. use 30 ul instead of 50 ul
Thank you all for your valuable suggestions. Though there is some improvement after dilutions, still I couldn't get reliable amplicons (only faints bands in the gel). Are there any other techniques on which we can rely upon?
Nested pcr is probably the best method but depending on how many cycles you are running in the first pcr it may be worth increasing the number of cycles of pcr. Are you getting a large primer dimer band amplifying at 40-80 bp size. thia can lead to low product amounts if it is happening ? Are the primers new or inherited from previous researchers in your laboratory?
Paul Rutland
Thank you for the suggestion, sure will try with this too. Nope, I am not getting any primer dimer and the primers I am using are results of previous works conducted in our laboratory.
If these primers are original primers inherited from a previous researcher ( rather than re ordered fresh for your research) then they may have been made up in water and one of them is depurinating. It might be worth titrating the primers...try 2x,4x and 8x as much primer and if the pcr improves rather than gets worse then have the primers remade fresh and dissolve in TE.
Safeena Majeed
reamplification of the PCR product fails most of the time. You should work on getting a good band in the 1st PCR.
Check if the template DNA is of quality and quantity. Did it work with other primers? Can you run a positive control?
Check if the primers are in good conditions. Often it is a good idea to buy new primers if you have doubts
Do a gradient of annealing temperatures.
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