Positive control contamination

[kleekris]

The purpose of our PCR assay is to detect presence of rodent parvovirus at the 420 bp region. When loading the acrylamide gel, we use a positive control (373bp) and a M13K07 bacterial phage (524bp) as an indicator for sample recovery. Recently, we've had 373bp positive control contamination in our sample lanes. When I try retesting the sample starting from master mix, the samples still comes out with 373bp contamination.
Our labs are set up such that we have 3 separate rooms: 1st room is the clean room where we perform aliquoting and DNA isolation; 2nd room is where we perform the master mix; 3rd room is where we spike our positive control tube, where PCR cycling occurs in the Thermocycler and where loading samples into the acrylamide gel occurs.
Anyone have suggestions of what the possible contamination routes are? I am stumped on this one...

[jonoave]

My guess would be the 2nd room - where you prepare the PCR reactions. That's where most contaminations are prone to happen: evaporation, splashes, carry-over etc.

[kleekris]

Well, that was my initial guess but if I retest starting from master mix using a seperate saved DNA sub aliquots, you would assume the 373bp contamination would be no more. That was not the case, 373bp appeared again which leads me to believe that the contamination is already in the isolated DNA which occured before master mix prep.

[jonoave]

If using a different DNA subaliquot and you still get the same band, then I suspect that the nature of your contamination is with the positive control primers. What is your 373 bp band? Is it a well-conserved housekeeping gene, that a homologous protein is also expressed by the parvovirus as well?

Note that all you can see is a ~370-ish band, and homologous or similar-sized bands which may differ slightly from your positive control gene can be amplified.

The next question is, which component that is contaminated? It could be:
1. Primers - Use a new batch of primers, preferably for all amplicons; target and postive controls.

2. Reagents. It is possible that the contamination could have spread to your reagents, e.g. Taq, buffer, Mg, water etc. I would suggest using a new batch of these as well.

3. Pippetes - clean out your pipettes, as it could be contaminated as well.

[kleekris]

My 373bp is the positive control and the 524bp band is my sample recovery band so I want to see a 524bp & 373bp in my positive control lane only and 524bp bands in all my sample lanes only but I am seeing a 524bp & a faint 373bp band.
As for reagents, we prep new master mix each time we start (they are all aliquoted for single use). Another factor I neglected to mention is that we have multiple analysts that start and pick up at certain stopping points in the assay such as isopropanol and next at the isolated DNA aliquot stage. Whenever I take the assay to DNA aliquot, there seems to be no 373bp contamination. If I bring the assay only to the isopropanol stopping point, the next analyst that picks up and completes the assay will have 373bp contamination. My suspicion is also technique of the analyst.

[ajithnandanam]

As you are not sure about the area of contamination, best thing you should do is that to use fresh set of buffers, taq and other reagents. make sure you change your gloves when entering one room to another.

Clean surfaces with Hypo solution: tables, door handles, fridge doors, etc.
Use: dedicated pipettes in each room,donot use same pipettes for mastermix preparation and sample loading.

clean your pipettes, use filter barrier tips.

contamination in PCR lab is a pain. clean everything and make fresh reagents before proceeding further.