smear for PCR products

[jamesgoat]

I did a PCR and ran a .7% gel to check the products. There was smear. I ran the sample before with the same condition and it showed some clear bands. Would it be the gel problem? Another thing I could think of is the temp. difference between forward and reverse primer. They are 5 C difference. Would increasing the melting temp help the PCR? Thanks in advance.

[LilKim]

So when you said "I ran the sample before with the same conditoins and it showed some clear bands" do you mean:

A.) You did a prior PCR with the same conditions (denature, anneal, extend. temps and times) and you had discrete bands.

B) You ran PCR product originating from the same reaction twice (on 2 separate gels) and saw bands the first time... and a smear the next time.

The gel isn't causing the problems ... something either happend to your DNA or your PCR needs to be optimized.

Please... explain a little-bit more so that we're on the same page.

- KIM

[jamesgoat]

A. decribes my situation the best. I am thinking to increase the annealing temp. to reduce some non-specific amplification. Not sure whether I am on the right track. The DNA is kept properly, so I think it should not cause any problem. Thanks btw.

[canalon]

Could you post a picture? So that we can have an idea of the kind of smear (all lane or potato shaped bands)

Degradation of your DNA sample is a possibility, because if the conditions were the same before, and yielded a sharp band, there is no reason that your PCR suddenly become non specific. Of course if increasing annealing temperature still works, that cannot hurt, but that is probably not the reason.
What do you call properly keeping your DNA?

Another solution would be a problem with one reagent. If for example you freeze your MgCl2 and do not vortex enough (1 minute at least) you may have invisble cristals that lowers the actual Mg concentration in the reacion and makes the PCR non specific.

[LilKim]

well if all of the conditions were the same... it's unlikely that changing the temps/times will improve the smear. It is possible something happened to the DNA (but if it was properly stored... that's probably not the case)

However, i am suspicious of your reagents ... has anything been left out too long? The first things to "go" are taq and (suprisingly) dNTPS. Also, check your concentrations of buffers and MgCl (make sure you're not adding too much).

Maybe you should try using another batch of PCR reagents (borrow some from a labmate) and dH20

Good luck!
- KIM

[oppox]

do u mean a smear as in a smear or one strong band with a tail?
Are u trying to fish for a gene?
Have u tried to purify the smear (pcr purification kit), is it getting better?

you can try increasing the annealing temp, you can try a touchdown pcr (havent tried it myself though). I always rund a reference reaction to see that its nothing wrong with the stuff im using.

[jamesgoat]

I tried to mix the rx mixture well and tried different conc. of DNA with hot start procedure. Higher annealing temp. was applied. A sharper band with smear came out from 200 ng genomic DNA sample. Some of these changes might help in the amplification. Hope everyone learns something from this sharing. Thanks a lot for the help.

[canalon]

200 ng template?!!! no wonder you have a smear! this is way too much....

[oppox]

yea begin to dilute it like 50 or 100 times or even more