Smear in PCR - (Apr/24/2006 )
Hello all,
I am trying to PCR up about 2kb region using forward primer of Tm 60C and reverse primer 68C. Both of them include restriction site and so they are long ones.
Usual PCR setting is
95C for 3 mins
95C for 30 sec
58C for 30 sec
70C for 2 mins
30 cycles
70C for 30 mins
I got 2kb band but at the same time there was smear all over so I increased annealing temperature to 60C. PCr product increased, as I see on the gel but smear remains.
What you guys would recommend, still increase annealing temperature? or just cut out the band and gel extract?
Using same DNA, I get clean PCR product for other genomic region, so DNA is not an issue here!!
Many thanks for all suggestions
The most frequent causes of PCR products smearing on a gel are excess template or too many cycles. I would guess that the primers you are using for this amplification work slightly better at your particular magnesium concentration and annealing temperature than the first set. Try reducing the number of cycles.
Do you think 30 cycles are too many?? I will give a try with 25 cycles.
How much of template do you recommend, in ng per microliter?
I got the same kinda problem in the past. In my case, it was an unappropriate amount of MgCl2. Try to get the salt concentration optimised for your primers. In my experience, increasing the amount will decrease your smear. If you are usign 1.5ul of MgCl2 50mM, try 2 and 2.5 and check how it works.
tfitzwater has already asumed that you have already done this.
I am trying to PCR up about 2kb region using forward primer of Tm 60C and reverse primer 68C. Both of them include restriction site and so they are long ones.
Usual PCR setting is
95C for 3 mins
95C for 30 sec
58C for 30 sec
70C for 2 mins
30 cycles
70C for 30 mins
I got 2kb band but at the same time there was smear all over so I increased annealing temperature to 60C. PCr product increased, as I see on the gel but smear remains.
What you guys would recommend, still increase annealing temperature? or just cut out the band and gel extract?
Using same DNA, I get clean PCR product for other genomic region, so DNA is not an issue here!!
Many thanks for all suggestions
I would suggest you lengthen the lower Tm primer to get closer to 68C (or shorten the other one a few degrees). Then do a touchdown PCR, if your cycler is able to.
Touchdown starts with an annealing temp about 5 degrees above the theoretical Tm (remember that ALL Tms given by oligo makers are theoretical!) for 4 cycles. Then every 2 cycles after that, the annealing temp is dropped by 1C, until you are about 10C below the theoretical Tm. Then do 10-15 cycles at that lower annealing temp. Works every time... assuming your Mg2+ is OK (which it should be unless you are very unlucky).
I am anyway using 2.5ul of MgCl2 for 25ul of PCR mix so I was thinking from the first point that I am using enough of Mg.
So I tried gradient PCR yesterday to check annealing temperature but upto 65C, I keep on getting band, so now the conclusion is from 58 to 65C, smear is present, them amount of product slightly increases at 61-62C.
I reduced amount of template DNA, as it was suggested by one of the list members..
I wonder what proportion of Taq polymerase to Pfu Taq polymerase ratio (in terms of units) is used by the members and how many units of Taq are indeed needed for PCR of 2 and 4kb DNA region?