I have had good, crisp 3 bands with a set of primers. All of a sudden, these same primers are giving a smear in water control, -RT and also +RT. Internal control is fine
following this and things were checked: primers were redrawn from stock, changed the water (which is fine with other people in the lab), changed the pipettes, changed the work bench, and finally gave it to another person to check.
The primers -working conc stored at -20 in TE and main 100micromolar stock at -20. The primers are just three months old.
Can anyone suggest what is wrong with my PCR reaction?
I would agree with Raul'd answer, but would add to check the sterility of your reaction tubes as you seem to be getting contamination in all samples and have checked the sterility of everything else?
You could try to clean up pipettes and then prepare the PCR reaction in an isolated enviroment like PCR cabin or laminar flow cabin, altough the filters of the laminar flow must be recent, otherwise the air will contaminate your samples.
I would agree with Raul'd answer, but would add to check the sterility of your reaction tubes as you seem to be getting contamination in all samples and have checked the sterility of everything else?
thanks a lot people for valuable suggestions
there is another thing to add.
three reactions from another person's master mix were taken: my primers+ other person's water , my primers + other person's cDNA, my cDNA + other person's primers
again same smear pattern repeated in first two samples.
i have attached the gel pic aith my query above
kindly go thru and help me
I agree with this point that your water or the tubes may not be sterile.. Do not touch any surface after using RNAase ZAP.... as i see from your gel image u r hving similar smear as -RT and +RT...however there is no smear in int ctrl... so try using sterile pipets.. n i guess this cn b problem with ur stock water... coz mayb tcontamination might be amplified by your primers...try using a fresh stock of water or borrow from some other lab.
I agree with Philip, check your reaction tubes.
Also the items used in running the gel, e.g. loading dye and TE too.
Looking at your gel pictures, it suggests that the contamination is most likely with the water or other PCR reagents (e.g. buffer). That's because the -RT control has a big smear, compared to the +RT and GADPH control. That would suggest non-specific amplification in the absence of template.
I can understand the frustration, things are working and suddenly they're not. :/
is it possible that because of the high specificity of the internal control primer, GAPDH is selectively amplified and therefore no smear appear as against the case of primers of my intrest?
is this possible?
Maybe this sounds weird but: stop trying to find an answer to the problem and work on the solution. Meaning: Change everything and I mean everything: different labspace or flow cabinet (at least new HEPA-filter), new filter! pipettips, buy nuclease free water (eg. qiagen: 129114), order new primers, dissolve/dilute in new nuclease free water, use previously unopened bag of certified nuclease free tubes, wear gloves (new box), use different pipetset, new previously unopened PCR ingredients. Then first perform a PCR with only neg. controles so no cDNA in the vicinity of your reaction setup. Clean? 2nd PCR with cDNA (preferably newly isolated). We have 2 laminar flowPCR cabinets with UV sterilisation. One for reaction setup, no template allowed. and another for addition of template. Succes!
I may be reading your results wrong, but it seems to me that your primer stock have been contaminated. This is a common problem if you are not careful and often work with the same material. If you are not yet doing this, Primers are so cheap nowadays I would just buy new ones. In addition, I recommend switching to barrier tips.
If your lab is set up for it, try working with radioactve labeling to see just how easy contaminants can spread from tube to tube.
Jesper
Just a recommendation. I know some people having little issues (like this one) with primers resuspended in TE, We usually use Milliq water instead of TE and works just perfect, no contamination, no other issue like that one.
Good Luck!
Alex.
people good morning.
yes i think my primers including the 100micro M stock is contaminated which am still unable to understand as I had never used it till yesterday .and some days back it was all fine
i had radiolabelled the primers to confirm there is no degradation.
so the final option was contamination .
ill order new ones.
thank you all for actively participating in this issue
Ramya
Check the plumbing of your water system. I have seen algal, fungal and bacterial growth in lines. It clearly looks like random amplification of DNA.
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