When preparing my mastermix or diluting primer stock, I take great care to maintain sterility by using filter tips and wearing gloves. I pipette fresh nuclease-free water into new, autoclaved Eppendorf tubes. After preparing the mastermix, I distribute it into empty PCR strips.
Next, I resuspend my colony mixtures by pipetting them up and down, and I add 1 µl of the colony mixture to 9 µl of mastermix for each sample. For the negative control, I add 1 µl of the same nuclease-free water instead of the colony mixture, then seal the tube tightly.
Finally, I add my positive control, which is prepared in a separate PCR tube (not connected to the strips, similar to the negative control). Once everything is sealed securely, I run the PCR with 35 cycles.
How can i still have contamination, and how do i prevent it?
One area of contamination is the inside of the pipettes used to load the pcr onto the agarose gel. Strip your pipettes down and soak in soapy water before reassembling. Meanwhile borrow a pipette from another worker and see if the contamination disappears. A thorough clean up with soap of your working area would help and change your lab coat in case contaminated electrophoresis buffer has got onto the coat and dust is falling off it giving a positive reaction in the negative control
The contamination hs probably come from buffer from the gel tank being transferred into your work area on gloves,coat or pipettes
It is possible that your mastermix stock get contaminated. It is highly recommended to dispense master mix in various tubes to prevent repeated freeze-thawing and its possible contamination.
I agree it could be either from master mix or water. For me it took a while to realize IPA was the culprit for bad RNA quality. I would try borrowing from another lab to rule out the reasons for contamination.
Do you mean the lower bands? If so, almost sure that it is "primer dimers" and/or unused primers. Try to 1) decrease primer concentrations in your reactions; 2) redesign primers.
It's not always possible to know where contamination originated. My advice, throw away ALL of your reagents and start over. That means toss the water, buffers, primers, etc. Don't reuse anything. Get a fresh box of pipet tips, new bag of PCR tubes, etc.
Do you have a designated gel-loading micropipette? If not, you really should. The easiest way to get contamination is by using the same micropipette for setting up PCR as you use for loading PCR samples in a gel.
Good luck!
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