6 replies to this topic

[Gunalan]

Hii all
I have been doing PCR iam geting a faint band in the negative control (with MQW) the product size is almost similar to that of the original product.

I tried few steps like
1.  Used new primer working stock
2.  used fresh autoclaved tips
3. used new (other) lab for making master mix under laminar air flow used their tips and pipets and reagents and dNTPs

still Iam getting the contamination what else can I try
Can anyone suggest me


Thanks
Gunalan

[lamaksha77]

It could be another of the PCR components that is accidentally contaminated with template, like the sterile distilled water or dNTP stocks.

Also, if you are loading the negative control right next to the actual PCR reaction on your gel, some spillover from the actual reaction (especially if the band if very adundant), could cause this....try skipping a well when loading...?

If it still doesn't go away, and the negative control well has only a very faint band, why not just go ahead with the rest of the experiment? You can play around with the contrast settings when capturing the image to make this band disappear if your experimental reaction has a very bright band  

[leelee]

lamaksha77, on 04 December 2011 - 07:54 AM, said:

If it still doesn't go away, and the negative control well has only a very faint band, why not just go ahead with the rest of the experiment? You can play around with the contrast settings when capturing the image to make this band disappear if your experimental reaction has a very bright band  

I think this is a really bad idea! You can't just ignore a +ve in your negative control, no matter how faint.
At best that is dodgy science, at worst it is academic dishonesty!

[Ameya P]

Gunalan,

You have not mentioned changing your water. I am not sure if you actually haven't changed it or is it just an omission while writing this post.
In case you haven't. looks like its your water that is contaminated with your template DNA.

TO confirm this, you could use the same aliquot of water but with a different set of primers. Your PCR product should now be the one you would expect with the new set of primers.

Please do not even attempt Lamaksha's suggestion, even if she meant it in a jocular way. You would not know when the contamination will come back to bite you.

A friend of mine whom I shared the lab with received a vial of contaminated plasmid when he started his project. All his attempts at cloning an insert failed miserably. After 3 painful (and highly valuable) months of his MSc project, he realised that he had started off on the wrong foot. He could really have made some good progress on his project had the previous noticed the contamination earlier and reported it.

So get cracking again, it should not take you too long.

[leelee]

gt_ameya, on 04 December 2011 - 09:49 PM, said:

Please do not even attempt Lamaksha's suggestion, even if she meant it in a jocular way. You would not know when the contamination will come back to bite you.

Ha! It didn't even occur to me that it could have been in jest, the winky face escaped my notice.......woopsy!

[mdfenko]

another component of pcr that is likely contaminated with dna is the polymerase.

you may have to rethink your primers.

Edited by mdfenko, 05 December 2011 - 08:09 AM.

[merlav]

Some times the problem is the instrument behind the pipet
1. Clean very well the pipets and if possible place them under UV