We are currently having problems with PCR amplifations of a ~200bp gene fragment. We cannot see any product after a 35 cycles amplification round and usually use a simple reamplification protocol: 1ul of our first PCR to a 20ul reaction. Resulting positive bands are not so clear so I wonder if you could kindly share your experiences with reamplification protocols. Thank you!
re amplification has a problem both with overamplification and the production of incorrect bands. 10 cycles of pcr is 1000 times more pcr product so often weak error bands become strong and also the greta over amplification of the right product means that product molecules anneal and you get a long smear of amplimer rather than a single band
I would dilute the first pcr product 1/1000 in water and set up identical pcr re amplifications but remove tubes at 14,16,18 etc cycles and you should get clean strong bands at a few different cycle numbers then the product will smear and become messy at too many cycles but it is necessary to tes how many cycles is the right number
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