6 replies to this topic

[sara.r]

I do some pcr on a purified dna fragment and I use maximum tm in pcr setting, but after electrophoresis on 2% gel I still have some smear below my expected band,I want to  to clone this fragment  so I need it in most purified form . I just dont know if extra bands are degraded dna or pcr by products (that are not expected in my pcr because i use a purified dna).
is there any sugesstions?

[mdfenko]

primer dimers?

[stylothecancer]

Hi sara.r,

Would you mind showing us your gel pic?

Thanks

[sara.r]

stylothecancer, on Jan 28 2010, 02:25 PM, said:

Hi sara.r,

Would you mind showing us your gel pic?

Thanks

hi
unfortunately I dont have a pic but my band is 1 kb and the smear in below it during the 750bp to 1kb region in 2% gel and it is not dimer primer.

[mdfenko]

could one of your primers be exhibiting non-specific binding? is there a site similar to the one to which the primer is made?

maybe you are experiencing some slippage?

can you gel purify the correct product or is the smear too close?

[swanny]

mdfenko, on Jan 30 2010, 02:07 AM, said:

could one of your primers be exhibiting non-specific binding? is there a site similar to the one to which the primer is made?

maybe you are experiencing some slippage?

can you gel purify the correct product or is the smear too close?

Wouldn't that start to make a discrete band, rather than a smear? Once you had 1 cycle of non-specific priming (which would be pretty weak), the next and subsequent cycles would be 100% compatible.

Sara, can you repeat the experiment with a few different starting template loads, in case the problem is there? Then you can show us the picture of the gel, which will really assist.

You can tell if the smear is from degraded DNA by treating some of the product with DpnI and running on a gel along with untreated product: if it is degraded DNA, the smear will disappear; otherwise, if it's amplified DNA from the PCR reaction, it won't.

[mdfenko]

slippage would not necessarily create discrete bands. slippage does not occur during primer binding. it occurs during extension. taq slips off and reattaches randomly (but usually near the slip off point) and continues on its merry way. this often occurs when extending sequences with a series of repeats or polynucleotides (poly-As, poly-Ts,...).