Hello everyone,
for a couple of weeks now i try to realize a PCR amplification of DNA extract from coral of the species Pocillopora.sp using ITS2 primer (internal transcribed spacer region 2)
for around 3 weeks now, i keep trying to realise the PCR but fail at getting negative Blanks for absolutly uknown reason.
the last PCR i did today just confuse me at the maximum.
here is the link to it https://drive.google.com/file/d/1kspwqAmiBQ5uAwCFNDg_x8jJa2x90qUq/view?usp=share_link
i won't describe everythink i did but i have been extremely carefull to clean everythink, use mask, lab coat and so on. I also used a laminar hood and placed all the material i used under an UV light for 30 minutes.
the first row of the gel is only blanks. i have prepared a master mix and haven't touched my sample at all. i just placed the master mix in one row of the plate and closed it with capes right away so there is absolutly no posibility for cross contamination.
so from that i just came to the conclusion that the contamination must come somehow from the material i use, even tough i take great care of cleaning everythink.
The second row goes as follow (sample/sample/sample/sample/space/blank/blank/blank/blank)
again, all my blank came out as strongly positive but then i don't understand why some of my sample came out negative since the first row tell me that the material is contaminated.
if my material is contaminated, although my sample is negative it should come out as positive
i am completly out of idea right now, i have tried basicly everythink that existed but can't figure out the problem.
if any of you has ever had a similar problem, i would really appreciate the help
We have to remember that 10cycles of pcr is1000 time more product and 20 cycles is 1000000 times more product. So even the tiniest amount of pcr product on dust,on the workbench or in a pipette will create huge amplification. You often get contamination by opening tubes or plate seals in your work area and tiny droplets are spread all over your area and the dust can get into the pcr reagents or tubes. Also if you run gels some product runs into the buffer then you lift the gel out of the tank and you get pcr product on your gloves. If you now go back to your work area then touch things then they are contaminated. Also if you use your pcr setup pipettes to load post pcr samples into the gel then some liquid gets past the filter into the pipette and that can then contaminate the reagents when you use the same pipette to measure out reagents.
I would put away or throw all reagents and borrow another researchers taq and all pcr reagents. You will have to order new primers but use new dilutions of the primers made up with another researchers TE or water and using their pipettes and tips not yours. Set up a pcr with 3 no template controls and one positive control or sample. Prepare the pcr using another researchers pipettes,tubes and working area.Use nothing of your own except primers. Also use their pcr machine. While you are running this pcr thoroughly clean your working area and strip down and clean your pipettes. Get new plasticware and clean your pcr machine.. The only sure way to get rid of contamination is to design one primer outside of your primer set but meanwhile lets try to get a clean NTC and a working PCR but it may take time.
I once flipped an entire 96-well plate of amplified PCR products near my bench. I cleaned, UV treated, tried setting up at a different bench, washed my lab coat, etc. But the only way to resolve the issue was to design new primers for the locus.
I'd also recommend getting a brand-new, never been opened, pack of PCR tubes and tubes for your primers. You really can't trust anything that you've already opened or used.
If all PCR reagents you use are new, it is likely that your contamination gloves, pipette tips, tubes, or the water you use, etc. is due. To avoid this, you must use everything new and make sure that all of them are sterile. My recommendation is to renew all PCR reagents and ensure that all materials used are absolutely sterile. I recommend using a positive control alongside the negative control.
Many thanks everyone for your reply, I will try to apply your advice
In terms of your specific case, if you are consistently getting contamination in your blanks, it is possible that the contamination is coming from your reagents or equipment. Make sure you are using dedicated equipment and avoiding unnecessary contact between samples. You may also want to try using a different lot of reagents or switching to a different brand to rule out reagent contamination. Additionally, you can try running a blank control after every sample to check for contamination.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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