multiple band on PCR - (Aug/04/2005 )

dear all..

i got plasmid after transformation (phew,at last!) and digest it using the same restriction enzyme i used to cut both vector and insert. and Thank God i got the right sizes and expected the bands.

But problem came after i further my analysis via PCR..i didnt get any desired band....the positive control showed multiple bands plus the right size. I assumed the annealing tempreture were not optimized enough.

What u guys think?any idea and suggestion?oohh yeah..i used the spesific primer for the PCR..

any idea and suggestion are all welcome..thanks..

Did you try to PCR from the digested product or straight from the plasmid?

You can try PCR from the plasmid using vector specific primers (like T7/SP6; M13F/M13R, etc), but when you come to size it remember that the vector primers will produce a product regarless of whether there is an insert or not (nice positive control for PCR conditions!). I dilute my plasmid sample (purifed using Qiagen kit) 1:500 in water, then use 2ul in a 20ul PCR reaction (or 1ul in a 10ul reaction) with standard conditions.

Hopefully it's just a glitch in the PCR. Include a positive and negative control to make sure that the reaction is working. And don't forget to pray to the PCR gods.

Nicole

I expect that you have put too much plasmid DNA in your PCR. Don't use more than 0.1ng of plasmid DNA and keep the cycle number under 25 cycles.

One thing that can happen if you are amplifying from a plasmid is "once around" PCR amplification. This can occur when using long extension times - you end up with a right sized band as well as a band the size of the vector + insert.

Daniel

Improved automated DNA sequencing traces

okay, i am not here to suggest you something, imstead i am facing the same problem, i am working on plant viruses, i designed 2.8kb fragment primer, but iam not getting same length amplification, i am getting multiple band with maximum 2.1 kb fragment.i tried everything primer conc. Dna input quantity, PCR profile, Mgcl2 conc, but it didnt work. even i designed primers twice, but all in vain.can someone suggest me something, which really help me

ususally multiple bands represent a problem w/ tm resulting in non-specific binding
look at the sequence to see if you have any similar binding sites, if the primer can half anneal to another spot in the sequence, then there is your problem. in that case you need to redesign ur primer.
otherwise make sure you have the correct annealing temperature, and you can also try lowering the extension temperature to 68 degrees for 3min instead of 72 for 2 min.

hope it helps