I am attempting to amplify a section of a plasmid that I have mutated to test for mutation. My primers are mutation-specific. My positive sample shows a band of the correct size (water, primers, dNTPs, buffer, taq, mutated template). My mutation control has no band (water, primers, dNTPs, buffer, taq, unmutated template). But my water control has the same sized band as my positive (water, primers, dNTPs, buffer, taq). I have done this experiment before with no issues, so I don't believe it is a problem with the PCR protocol (annealing temps, number of cycles, etc). However, I can't believe it is a contamination problem because I don't get this same band in my mutation control. I have tried new master mixes three times, double checking each time to make sure I didn't make a loading error. I make the master mix in a separate room using filter tips. Any advice would be appreciated.
Dear Emma
There are no need to change PCR reaction protocol, because without any template no amplification should be occur in your negative control (using only water and no any template). Coming the same size band in your negative control it conforms that there are mutated template which amplified during PCR. There should be contamination of template of mutated DNA in your reagent which you are using for negative control. Please try to find out through using the new set of all reagents for all PCR (Muted, Unmutated control and Negative control) which you are using in your PCR.
With best wishes
Kamlesh
From your explanation one this is clear that
1. No band in mutation control so there is no sort of contamination in PCR components
2. +ve control reaction works well no issue with PCR conditions
Now possiblity remaining is
1. Handling problem: Use of same tip of control DNA while adding master mix in +ve and then -ve reaction but prior to muation control
But what is thes results in case of test samples?
it does look like the water in the water blank is contaminated as all of the other reagents are common. So is the water that you used to dilute reagents and to make up the volume in the pcr reaction mix exactly the same as the water added as a water blank.Try amplifying a range of water blanks with water samples from other labs,other researchers reagent kits etc against your positive reaction water. It is worth remebering that sterile water is no good for pcr as sterile means no living organisms but dead organisms still have dna so maybe also dnasing a water sample from your blank against an unmodified sample might be worth a try
Sachin: I agree, it is probably a handling problem on my part. I am redoing it today, being extra careful with all reagents (brand new), cleaning my pipettes, etc. I am also placing my water negative in another pcr machine to avoid any cross-contamination that might have happened inside the machine previously.
Paul: for my "water" negative, I actually don't add any water, so it is a misnomer on my part. I just aliquot my master mix to 0.2ml tubes, and one tube I never open again until I am ready to load the gel. This I call my water negative, even though it is a control for my master mix. I do use ultrapure water from a company, that my RA aliquots into 1ml samples for the rest of the lab to use (so we don't contaminate the source bottle), so I am pretty sure the water is clean. Thanks for the tip about the sterile water though, I had not considered that dead organisms would, of course, still have DNA!
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