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peace upon you

peace upon you - PCR - Polymerase Chain Reaction Forum

peace upon you - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 01-25-2013, 12:10 PM
Pipette Filler
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Default 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 ???
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Old 02-02-2013, 05:29 PM
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Default 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.
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Old 02-03-2013, 01:15 PM
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Default 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
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Old 02-14-2013, 06:53 PM
Pipette Filler
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Default Re: peace upon you

thanx for your welcome
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Old 02-14-2013, 06:55 PM
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Default Re: peace upon you

thanks for response .
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Old 02-14-2013, 06:59 PM
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Default Re: peace upon you

that help me a lot. thanx for all of you
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