The long smear in negative control - (Dec/15/2005 )

Hi everybody!

I'm a new member in this website and I'm new in doing PCR too.
I want to amplify the 1st PCR product from genomic DNA extracted from human blood.
After run the PCR and checking the product in gel electrophoresis, there is a long smear in negative control (use doubled distilled water instead of DNA template) as you can see in attached file. What should I do to solve this problem?

Thank you very much

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Well it looks either like genomic DNA contamination in your premix or in the reagents as you know negative contrôl should show no band.

I don't know how skilled you are in pCR so don't be upset this are general remarks

1/ Try to work only with aliquoted reagents if contamination appears you can trash the aliquots
2/ Assemble premix reactions and pipet DNA with a separate pipet at the end after aliquoting your premix in each tube

3/ After amplification use special tips (filters tips) to avoid contamination of your pipets, load your sample each with a fresh tip that way you can avoid carrying some positive signal from the previous sample

In your case I would vote for a contamination of one of your reagents

Pesji

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I agree with Pepji. Is the ddH20 used in your negative control the same H20 you use for your PCR reaction? If not, start there, and use the same high quality H20 you use for your other reactions. Good luck!

LabGirl

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In your first gel, the neg. does not look like something that has been amplified by PCR, at all. What's the largest band of your ladder; a few kb?
The major part of the neg is larger than that, it looks like partially digested large size DNA, and there is much of it. Too much to be some contamination in a dd water. It has produced what I call a "blubb" (too much DNA too make a sharp, fine band) which indicates that it's overloading the gel capasity, which is roughly a few hundred nonograms per 5 mm x 5 mm x 0.75 mm well in a 1 % gel. Try reading about gel capasity in a lab manual, if you are interested.

In your second trial; how much of the former neg did you add? It looks like a diluted version, still don't look like its amplified.
The new neg however, does look more like it could be something amplified.
You do have some major contamination. Problem is that most of the bands in your neg, do not occur in your samples. It might be because the contamination is in your water, and that there is more water in your neg. But normally one would not add that much more water in the neg, to account for such differences seen in your gels.
Try checking what the difference is, and if it's enough to account for the intencity difference of bands og neg bands.
And never, ever put anything in the neg that do not occur in your samples. When filling in with water, make sure it's the same tube you used for your real PCR reactions.

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Just one more suggestion to add - are u getting some contamination from yours or other peoples pcr products? cos i agree with gerd there seems to be too much DNA there for it to have come from water or a reagent?

using the same reagents/pippetes/ddH20 etc can increase yr chances of that... i would also give the area u set up yr reactions in a through clean up...

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