hi!!

it has been 3 weeks that i did PCR for 3 strains with different type of primer which make it 3 reactions n the gel shows a perfect band. but when i sent my pcr product for sequencing, it always show very very low peak n it dissapear at the end. i did change my primer n it is a brand new primer. i also change the temperature setting for annealing etc.. and it drives me crazy as my supervisor keep on asking for the results.

p/s: i did a MLST for Staphylococcus aureus. identification for Staphylococcus aureus needs 7 primers. for these 3 strains all 6 primers showed a good sequence xcept for 1 certain type of primer. for example:

strain
164 primer glp-F
729 primer aro-F
733 primer yqi-F

i juz need your opinion about this as im not very experience in PCR and DNA sequencing. i did sequencing for other strains n primers n it result doing just great!!

thanks a lot!!

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hello,
may be you have some contamination in your sampler/pipitor or may be you have proble in your pipptting practice and as the consequece you may unintentionally contaminate your sampler/pipette.
good luck
meh

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Hi, I have a similar problem.
I am doing ChIP experiments. THe first time I did it, PCR and everything worked very fine but now I can not reproduce it at alll.
What I get after PCR is a smear starting from the lanes
( I am also working at another project with BAC plasmid (197kb) and doing PCR from this plasmid).
Can one have a PCR contamination as a smear or is it rather several distinct bands?
Thanks

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Hi all,

I'm having similar difficulties to those mentioned in this thread. I'm running a PCR reaction using Taq polymerase purchased from invitrogen to amplify cDNA synthesised from RNA extracted out of bovine oocytes. I' using both Bov 7/11 primers, and H2A primers. I'm getting positive bands in my negative controls in some instances. Before the Christmas break I isolated the problem to some of my reagent stocks, so I obtained fresh stocks and repeated the same experiments. Since then some of my negative controls are clear, and some of them are contaminated with positive bands.

since returning to the lab I have taken delivery of fresh stocks of PCR buffer, Taq polymerase, MgCl2, and dNTP. I have also received fresh stocks of Bov 7/11 and H2A primers. I'm not convinced the problem is contaminated reagent stocks, since by now I've changed every single stock solution to no avail.

It may simply be that my technique is flawed, this is my first time running PCR reactions, but I've been running them for over a month now, and some of my negative controls are coming back perfectly clear.

In addition in the last 2 weeks I've moved my work into a new hood, this is much better outfitted compared to the hood I was using. People have mentioned exposing my mastermixes to UV before splitting them between my sample and control tubes. Will this damages any other components such as dNTPs? Would it be worth my while UVing all of my reagent stocks?

To further complicate matters I'm also running PCR usign a proofreading platinum Taq polymerase also purchased from invitrogen. I'm obtaining smears in many of my wells, but my negative controls are coming back clean. I guess the only significant difference is that I'm using reagents from a completely different kit so presumably they're contamination free. Does anyone have any other suggestions as to what might cause smears in proofreading polymerase enzymes?

Thanks in advance!

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Is it possible that the BSA found in many of the buffers and enzyme mixes is contaminated with bovine DNA?

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possible? yes. likely? no. the companies use the purified bsa fractions that are certified for molecular biology use (or so they say).

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It seems the contamination was coming from our stock of dNTPs, I've since made up a fresh working stock and my NTCs are clear now.

The smear problem persists with the proofreading polymerase hwoever. I'm working on the basis that a smear is the result of PCR product containing amplified fragments whose differences in terms of base pair ength are too small to be resolved by horizontal gel electrophoresis using 1 % agaroe? That being the case I'm assuming these fragments are the result of a more efficient proofreading polymerase synthesising DNA strands following non-specific and partial primer annealing events?

Assuming that's the case I'm going to run a proofreading PCR using product from a PCR run using regular (non-proofreading) Taq polymerase. I'm hoping that the excess of specifically amplified gene product will reduce non-specific primer binding, with a subsequent reduction in the spectrum of dna fragments sizes being produced.

Does that make sense to anyone else or am I compeltely off track?

Also I'm thinking of reducing the amount o primer I'm using for my proofreading PCR to reduce non-specific annealing......thoughts anyone?

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The smears can be due to different concentration of ur primers in the reaction so they are ssDNA, try to get an OD and reajust the amount ur adding in the tubes.
Hope this helps!!

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my 2 cents ... though this may have been mentioned already
1) if you are using platinum Taq, be sure that the initial denaturation step is long enough (ie: 5 minutes at 94 degrees, before you go into the regular cycles)
2) my experience is that smears mean the annealing temp is too low: try raising it 2 or 3 degrees
3) you may have to fiddle about with the Mg concentration
4) is your Taq able to handle the length of the sequence: that is, if you are amplifying a long sequence you may have to go to something like "Expand" (Roche)

Best of luck!!

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These are the only two thigns I haven't tried by now, might give them a shot sunday. Thanks mito.

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