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PCR problems with 1,7kb fragment - (Jun/29/2006 )

I need to amplify 1,7 kb fragment from a plasmid. I confirmed the insert using restriction enzime. I use two primers of 24 (Tm= 68,8° revers) and 23 (Tm 59,8° forward) bases with this conditions:

Buffer 2 uL (1x)
dNTPs 5.7 uL (0.2 mM)
Mg++ 1.6 uL (2mM)
Primer F 0.5 uL (0,5 uM)
Primer R 0,5 uL (0,5 uM)
Taq 0,1 uL (0,5 units)
H2O csp 20 uL

95°C 5 min
95°C 1 min
72°C 2 min (Anneal and extend)
repeat 30 times
72°C 5 min

Using this protocol I didnt get the fragment, so I repeat the PCR but using 72° by 3 minutes. Again i didnt get the fragmen.

Should I use a lower Temp for annel?... I mean, something like 64°C
Sholud I increse time of extend?
Should I increase Taq, dNTPs or primers amounts?

Thanks for your help.


The difference in Tm between the two primers is 9 degrees. I think it's a bit too high. Maybe you should try with 64 degrees for the annealing. The remaining looks ok in my opinion


An annealing temp of 72 is way too high. Your lowest primer has a Tm of 59. I would try 57-58, but you might be able to get away with the low 60s.


try annealing temp. around 60C.

how much do of template do u add to ur mix ? try 100ng

U could also try different Magnesium conc. , they can make a difference as well.

good luck !!!


I would try 58 or 60°C of alignment. 100 ng sound too much for me!!!!
I think 1 min 30 seg of time extension would be enough.

-aztecan princess-

anealing temperature is around 50 to 60max.
extention temperature 72 for 1.30 to 2 min is good.
you must optimise your anealing temperature.


The enzyme of LA taq produced by takara may have good efficiency. you can try.


if you can, I would try a Ta gradient from ~54 to 64

although I also think 9 degrees is awfully different. you may struggle to get specific product at a Ta low enough for the rev primer to bind


What polymerase are you using? PFU needs a longer elongation time. Another good trick is to increase the concentration of MgCl2 to aroud 3mM. This sometimes makes the reaction work better. Especially if using cDNA as your template.