pcr smear

[pooja pandya]

Hi all...M currently working on Sunflower genes. I am encountering a big problem of DNA smear and in-consistent bands since last 4 months in my Nested PCR. I have tried everything mentioned above but no improvement, though i havent tried with diluting or making fresh DNA samples. This is because the same DNA samples give tremendously good results with other gene and primers. My forward primer Tm is 63.0 and Reverse primer Tm is 65.0, I have tried with gradient of 55 to 60 degrees, but no amplification. should i go lil higher on annealing temp?
I have exceeded my project dead-line...kindly suggest.

[pooja pandya]

hello

[hua052011]

Originally Posted by saly

smear may be means som hydrolysis

I do not understand what you said about

[hua052011]

Hi,

Thanks very much for this comment. It help me to think about my ideals.

Tks again and pls keep posting.

[davidflora]

Try to get the salt concentration optimised for your primers.

[Jenniferayt]

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[saly]

Originally Posted by bhaktamehul

Hi,
i want to know what are the causes of pcr smear. coz i have been having some smear from last one week , but early it workd great... i even tried new dna samples, made changes in annealing temp and time and also used same primer from other lab(where it is working),,

so i just want to know what can be the causes of smearing , so that i can sort out problem...

mehul

Hi
The first reason for smear is DNA which was degradation , must prepare new DNA fresh with good concentration and try again .
cheak the primer on the agarose gel
good luck

[saly]

Originally Posted by pooja pandya

Hi all...M currently working on Sunflower genes. I am encountering a big problem of DNA smear and in-consistent bands since last 4 months in my Nested PCR. I have tried everything mentioned above but no improvement, though i havent tried with diluting or making fresh DNA samples. This is because the same DNA samples give tremendously good results with other gene and primers. My forward primer Tm is 63.0 and Reverse primer Tm is 65.0, I have tried with gradient of 55 to 60 degrees, but no amplification. should i go lil higher on annealing temp?
I have exceeded my project dead-line...kindly suggest.

Hi
The first reason for smear is DNA which was degradation , must prepare new DNA fresh with good concentration and try again .
cheak the primer on the agarose gel
good luck