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may84 01-25-2013 12:10 PM

peace upon you
 
im working on 3 human gens about ( 200 ,300 , 400 )bp .

my samples genamic DNA from blood .

my supervisor dilut the primers to 100 pmol.

i use Accua pwoer primix = 5 microliter

total reaction volume = 20 microliter ( 9 microliter DNA ,, 1 microliter primers )

program : 95 c ..5min

95 c .. 1min
55 c ..1min
72 c ..1min

72 c .. 7 min

35 cycles (thermal cycler pcr )


at first the pcr is ok and the product perfect .... then primer dimer result and no product . what's going on ???

deepak1228@gmail.com 02-02-2013 05:29 PM

Re: peace upon you
 
Hey there,
You can try two things here:

1. Reduce the annealing temp. Rather setup a gradient.
2. Add only 1 primer, wait for about 5-6 cycles and then add the second primer.

osama al refa'e 02-03-2013 01:15 PM

Re: peace upon you
 
hi

since it work at the first time :
you have to check the step
the amount of the genomic DNA you have
try to increase the annealing tem 58
really you have check the technical error

good luck :)

may84 02-14-2013 06:53 PM

Re: peace upon you
 
thanx for your welcome

may84 02-14-2013 06:55 PM

Re: peace upon you
 
thanks for response .

may84 02-14-2013 06:59 PM

Re: peace upon you
 
that help me a lot. thanx for all of you


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