Annalucinda

hi all,
I have done PCR using a universal primer and eluted the PCR product from the agarose gel using Nucleospin kit. Eluted pcr product  on agarose gels is showing multiple bands on gel electrophoresis.
the PCR product of mine only showed single band but after elution am getting multiple bands...why am getting this multiple bands after elution?
I have repeated this elution using QIAGEN kit, i have also tried with different primers, and also modified the elution kit method (cooled the sample to reanneal after incubating at 50ºc for melting the gel) ...but still am getting multiple bands after elution. from the multiple bands of eluted product i have also cut the desired band and eluted that,but still i got multiple bands.. please give me a suggestion to solve my problem....
thank you in advance..
-anna.

First of all, when you are describing a reaction set up, you need to specify concentrations, not volumes.  For instance, if you are using a standard 10mM dNTP stock, then 7 uL of 10mM dNTPs is way too much.  Usually 1 uL of a 10mM dNTP stock in a 50 uL reaction (0.2 mM dNTP final concentration) is used.  The same with primers.  Each polymerase is different, but a typical range would be 1 uL of a 10mM primer stock in a 50 uL reaction (again, 0.2 mM final concentration).  Lastly, your template (miniprep DNA) should be at a final concentration between 0.1 nM and 0.5 nM, which means that for an average 3000 bp plasmid, you would include about 50 ng of DNA in a 50 uL reaction.

All of these concentrations can affect how well your PCR works.  Lastly, your PCR cycling conditions are also critical.  As Adrian K suggested, you could try gradient PCR to find a good annealing temp, but in case you don't have a gradient PCR machine, I would just try this PCR program:

95 degrees, 2 minutes
----------------------------
35 cycles of:
95 degrees, 30 seconds
50 degrees, 30 seconds
72 degrees, 1 minute/kb of PCR target
-------------------------------------------------
72 degrees, 10 minutes
4 degrees, infinite

This program may generate some non-specific products, but you should at least get the band you are looking for.  If you need to get rid of backgroud, you can always try raising the annealing temperature (from 50 to 55 or 60) AFTER you can actually get a product.

Best of Luck.