peace upon you

[may84]

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]

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]

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]

thanx for your welcome

[may84]

thanks for response .

[may84]

that help me a lot. thanx for all of you