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pcr trouble solved but I cant seem to understand why


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#1 MaggieRoara

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Posted 26 October 2009 - 06:17 PM

I had some pcr problem for about two weeks. On the positive note, I have solved the problem. I merely repeated the PCR again and again, each time changing each of the components of the reaction, noting when the control worked and when it didnt. At last I narrowed it down to not adding enough template. However here is the part I dont understand. My control seems to work with 10mM of template, but my template needs 100mM. With 10mM my template doesnt work. I also see a blur bans thats well below the 500bp mark on my ladder (neb 1kb ladder).

My control is just my lab mate's template and primers with all other reagents common with my reaction. Her pcr product is 500bp mine is 1.1kb. her primers are different from mine and her plasmid is different as well.

Please can someone help me?


Thanks

and oh I measured all my dna with nanodrop so it should be accurate.

#2 swanny

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Posted 26 October 2009 - 08:53 PM

I had some pcr problem for about two weeks. On the positive note, I have solved the problem. I merely repeated the PCR again and again, each time changing each of the components of the reaction, noting when the control worked and when it didnt. At last I narrowed it down to not adding enough template. However here is the part I dont understand. My control seems to work with 10mM of template, but my template needs 100mM. With 10mM my template doesnt work. I also see a blur bans thats well below the 500bp mark on my ladder (neb 1kb ladder).

My control is just my lab mate's template and primers with all other reagents common with my reaction. Her pcr product is 500bp mine is 1.1kb. her primers are different from mine and her plasmid is different as well.

Please can someone help me?


Thanks

and oh I measured all my dna with nanodrop so it should be accurate.

what are the details of your reaction mix? What kind of template are you using?
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