I have sterilized the working lab, autoclaved the plastic wares and have also changed the reagents like buffer, MgCl2, DNTP, Taq, water etc. I am generally working in Laminar flow hood.
Primers should always be prepared and diluted in sterile autoclaved water for PCR use. Try to use a fresh aliquot of water and primers.
Most people ignore the fact that one of the major source of contamination is from aerosols. If you are using highly concentrated template, even a miniscule aerosol may have sufficient DNA to spoil a subsequent no template PCR. DNA from Aerosols can also enter the vaccum generated by the pipette and stick to the inner walls. When they dry, they flake and fall to the solutions/PCR especially in the force of the pipetting out. If highly sterile work is must, a separate pipette should be reserved for such applications. Also using pipettes of appropriate capability, which have not too much suction power will generate less aerosols. A better approach is to use block tips/filter tips. This is extremely essential in sensitive PCRs. I guess you have taken care of the routines such as clean reagents and workplace, as mentioned.
change pipette set also dont let the flow on only sterlize by uv before putting pcr
Dear Pravin,
Do the other samples have same size bands as negative control has? Then avoid those bands for routine work. For sensitive work, please change the aliquot. If your colleagues use PCR reagents from the same source, please you make a separate box for you. Sometimes colleagues incautiously contaminated the PCR reagents. I faced such type of problem.
And, I agree with Raman Ratheesh
Dear Pravin,
Please try following:
Use TE buffer for primer stock preparation (master and working)
Use dedicated laminar for primer stock preparation as well as master mix preparation.
Use dedicated laminar for template addition.
Keep UV of the laminar as well as room ON for 15 to 30 min then start your work. Also use RNA Zap for cleaning surface before and after use.
Thanks
Dear
Please try the following measures;
- dont exit any implements/apparatus from laminar even for a second.
- always use ethanol (if your material is not sensitive to ethanol) to wash hands and gloves as well as any other tools before handling your materials.
- dont open your material out side laminar and dont handle your material without gloves.
Thanks
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