PCR using DNA eluted from agarose gel as a template

[dawitgirma]

Hi all,
I have a problem with my gel eluted 4kb DNA fragment PCR amplification. After Gel elution I got around 40ng/microliter of DNA concentration and I use 2 microliter out of this for PCR. But I always get a bad smear on my gel picture instead of a specific band. Can anyone help me on how to fix this problem?
My PCR programme is as the following,
1. 94°C - 2 min
2. 94°C - 45 sec
3. 58°C - 45 sec
4. 72°C - 4 min
5. Goto step 2 and repeat 29 times
6. 72°C - 7 min
7. 4°C END
Thanks in advance,
Bests,
DG

[Drekketh]

Well, at the top of my head I can pull a lot of troubleshooting regarding DNA gel extraction, DNA tempate concentrations, primers and PCR cycle conditions.
For starters I would recommend you to use lower DNA template concentration. You use 80ng per PCR reaction, lower it as much as possible and re-run you DNA extracted in another gel (just a small quantity), in order to check the intensity and putiry of the band. Once run and your band is crystal clear, use your extracted DNA in a lower concentration.
Btw, what is the company of your DNA extraction kit?

[dawitgirma]

I used Qiagen dna extraction kit.

[jonoave]

I use a QiaGen gel extraction kit and it works fine for me too. As Drekketh suggested above, the main culprit is likely a high concentration of template.

What I generally do is a dilution starting from 20x. If smears still appear, I increased it to 50x and finally to 100x. Usually by 100x the smears would've disappeared.

[Phototoxin]

I concur, mix up some dilutions and run them and see if it makes a difference.

[SebaQ]

Agreed... it's likely to be the DNA concentration. Serial-dilute your DNA and re-run your PCR with the dilutions. The more diluted the DNA, the cooler the band you get =D!

[Alicialsw]

Will a high concentration of template inhibit the whole PCR reaction and thus no band formation?

[SebaQ]

Originally Posted by Alicialsw

Will a high concentration of template inhibit the whole PCR reaction and thus no band formation?

Totally. PCR works better the lower the DNA concentration, 'cause polymerase is regulated by product concentration.