I am currently using Q5 polymerase from NEB to amplify a protein of 2.5 Kb. I have designed the primers to target the 21 BP that are compliment to the DNA sequence , the restriction sites are added before the complimentary sequence along with a 2 BP cap for frw primer and For reverse I made 45 bp primer with 25 bp overhang and 20 bp complimentary sequence . The primers have been checked for hairpin formation and dimerization. I am using a standard set up, for the PCR reaction 98C initial denaturing for 30sec, then 30 cycles - 98C 30s, 65C 30s, 72C 1min, then final extension at 72C for 5 min. I seen an accurate faint band at 2600bp and many non-specific products. I appreciate any thoughts you all may have and please let me know if you need any more information.Thanks.
Frw Primer
GC CONTENT = 51.7 % Hairpin = -0.78 kcal.mole-1 Self-Dimer = -7.31 kcal/mole Hetero-Dimer = -4.74 kcal/mole
Tm= 67°C
Rev Primer
GC CONTENT = 59.1 %
Hairpin = -1.9 kcal.mole-1
Self-Dimer = -8.02 kcal/mole Hetero-Dimer = -3.29 kcal/mole Tm= 64°C
Anneal = 65°C
Dear Rishi
the question is: what would like to do with if afterwards? Cloning in a plasmid? whith which approach?
because if it is True that you have a lot of aspecific, is also True that those aspecific are far away from your bands therefore you can try to perform gel extraction and purification of your pcr fraction to clean it with a kit. of course the yields of those kits are not amazing, so in this case it is better if you perform higher pcr volumes with the same protocoll and purify and extract an high amount of sample.
however if you would like to optimize the pcr you can try to perform a touch down protocoll where for example in the first 10 cicles you use higher annealing temperature which decrease
eg 70c at cicle1, 69.5 at cicle 2 - 69 at cicle 3 and so on up to 65. 5 at cicle 10 and afterwards 25 cicles at 65 ad you already done.
i wish you have a pcr machine able to set up automatically this programm.
orherwise you can try:
-repeat the pcr increasing the 2nd temperature from 65 to 68 for example and are of your specificity increase.
- test one other taq. ( eh clone amp hifi takara or kapa hifi roche). some times different taqs provide very different results.
just a final comment:
in my experience, with aspecific with so different size: with some cloning approaches ( eg PIPE cloning) if you have on your hands a good colony screening approach, you can try to go on also with this pcr because if it is True that probably you need to screen several colonies to find one good, is it
also True that also pcr optimization is not obvious
your impurities are so different that with pcr colony you can easly distinguish right colonies from wrong colonies.
So in case you use standard enzyme cloning i suggest to you to purify the pcr from gel and in case you use PIPE cloning, in-fusion cloning or similars before optimize the pcr try to go on with this pcr and see what you will obtain.
of you would like some information about PIPE cloning and pcr colony screening protocol you can find it on my blog; https://proteocool.blogspot.com/?m=1
good luck
Manuele
1. I am trying to do overlapping pcr.
2. There are 2 protein segments one has overhang at 3 prim and the other one has a overhang at 5 prim. With the help of the frw primer of this protein and rev primer of second protein i can amplify the whole segment at once and put it in a plasmid by Restriction digestion and ligation.
Hi there,
You may increase the annealing temperature (Tm+3°C or even 2-step PCR) or add DMSO to increase the stringency of annealing. An other point concerning the extension time: NEB gives 20 to 30s per kb for extension with Q5, so you may extend extension step in order to be sure the 2.6kb sequence is properly amplified (I would try 90s instead of 60s).
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