I extracted HBV DNA from serum (frozen, stored at -80 degree). I used Qiagen's HotStarTaq Plus PCR Kit to amplify the region encompassing the preS1, preS2, S gene. the product is 1373bp long.The PCR recipe is as following:
Taq Buffer 1x
dNTP 200μM
forward/backward primer 0.3 μM
MgCl 2.25 mM
Taq 2U
template DNA 5μl
[reaction volume = 50 μl]
Thermal cycle: initial denaturation at 95 degree for 5 min. then 40 cycles of 95 d for 30s, 49d for 1 min. 72d for 1:30, at last 72d for 5 mins as final extension
I follow this protocol for months and it was giving satisfactory result. All i get is smear now, even when I use template that gave band before.
Does the smear result from low annealing temp? In the past i used the primer stock in lab (unsure of what supplier). After they were used up, I bought new stocks from IDT and the Tm of the primers are 53 and 52.8 degree, similar to the old stock. i just checked the analyzer on IDT web and it said under 2.25mM MgCl the Tm of the primer will become 61.9 and 61.4. Does this mean I should run PCR at 56 degree instead? 49 degree worked pretty fine in the past with the amount of MgCl i added. I am puzzled now.
I replace all the reagents already including water but the problem presist. I have stucked for a month now but neither my collegues or supervisor got a clue. Please help :(((
Try reducing your MgCl2 to 1.5 mM, 2.25 mM is a bit higher. The smear can also arise due to excess amount of template or primers. Try reducing your template to 2 microliters or dilute your template. Also try reducing your primer conc. to 0.2 micromolar. In your case I think its the MgCl2 conc.
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