Hi Julieta, water controls? I would recommend you to use always fresh autoclaved water in reaction tubes. Use it once!
And aliquot your primers etc.
Use filter-tips.
Best, Nadine
Hi. try to use aliquots of each product, autoclaved DDW, work in a sterile environment, filter tips or sterilized tips etc.
Bye
Hello!
I agree with the things said above.
In addition, you can use a bench top mini UV-light cabinet, in which pipettes, tips etc. are placed before you start, UV-exposed for 30-60 minutes in order to destroy potential DNA contaminations on surfaces of whatever you gonna touch.
Alternatively, use a cell culture cabinet with UV light. If you don't have either, try to find a place, where nobody isolates any DNA.
Keep your template DNA as far as possible from all other reagents. First pipette your mastermixes and put them into the reaction tubes. Then add water as controls. And only then handle the template DNA.
Also, change gloves often.
Use pipette tips that are longer than your tubes so that the pipette itself does not touch the inside of tubes.
I hope this helps!
One question in the beginning: Do your negative control primers always give a signal or only sometimes? If the first is the case, this might be due to the formation of primer dimers. Whats the size of the product?
Are you sure, your primers are free of contamination? If this is the case, you will not get rid of the problem with other methods. Try making a fresh wirking stock (preferably at a different working place with other pipettes and fresh water).
Using filtered tips might also help, although they are more expensive than normal tips.
Use freshly autoclaved tips, use nuclease free water, and make sure that while loading in the gel there is no overflow into the next well...
Thanks for the recommendations, I already started to use some of them ( filter-tips, use freshly autoclaved tips and whater) , but all of them are really helpful! I'll try them! About the formation for dimer primers, no I don´t think It could be. Also I would like to ask you, How can I prevent interfered bands using BSA in my reaction mix?
BSA generally "soaks" up various sample-related inhibitors in the PCR/qPCR - not sure if it is thought to affect banding. BSA has been lauded for use with archival, even archeological samples subjected to qPCR as well. Betaine and DMSO (and several other agents) are normally used to affect PCR specificity - but those may already be in your mix (not sure; proprietary info in many cases). If you read up on Betaine, it has an interesting story - possibly helpful to you. On contamination, two words: Andrew Wakefield, Andrew Wakefield, Andrew Wakefield (wait - that's 6). ;] AKA: Having a separate, isolated area to set up your reactions is very important. There are most likely trillions upon trillions of unaccounted for plasmids and amplicons afloat and adrift in many research facilities (not to mention the plethora of left-over/sloughed biological components/DNA found amid the dust of every room). Imagine the unusual biodiversity of designated land fills at this point... housing all of our orphaned reactions as part of the responsible rituals of disposal.
Hi Julieta
Every step and component of your PCR can introduce contamination. Another word of warning is, be extra careful to ensure your negatives are really negative if you're sending your samples for high-throughput sequencing. I don't know how much of your negative you run on a gel, but for this I'd load it all, to ensure there really is no contamination.
The important things to remember for contamination-free PCR, some of which others have mentioned are:
1) have a separate area for PCR, and if at all possible use a UV cabinet. In this you can have pipettes dedicated to PCR, thus ensuring they are not contaminated during other uses, an also your tips (filtered of course).
2) the water is important. Yes you can autoclave your own, but there are some reports of DNA surviving in the autoclave and this may be a source of contamination (you should find these papers easily enough through Google). I generally buy in sterile water and aliquot it into fresh, sterile, DNAse and RNAse-free tubes. I then use each tube only once.
3) UV everything before you start, i.e. tubes, gloves, tube rack etc, even reagents (buffers, MgCl2 etc, though obviously not the polymerase!
4) the length of the tips, as Alexander mentioned, is an obvious but important point that is often overlooked
@Laura: A few comments to your post:
1. A seperate PCR working place will not necessarily solve problems, because you (or other people using it) can contaminate the pipettes when they let the handles snap up. This has happened in a lab I worked and it took a while to find it. Since you are working with your target DNA there, this can generate a lot of confusion.
2. Water: I would never use autoclaved water for PCR (especially not, if the same autoclave is used for autoclaving the waste in the lab). If it is not completely destroyed in the autoclave, so you can PCR up things from it. We usually used sterilized medical water, that was aliquoted into small portions, when we opened a new bottle. So you can discard the stuff if you doubt its purity.
3. UV-light: I find UV-light over-rated. First, its bad to all kind of plastic ware which is used more than once (this includes the shafts of the pipettes). Most UV-light has either not enough energy to break down DNA completely or it is not applied long enough (think about gel extractions on a UV table which still leads a lot of usable DNA). Then these hoods tend to be crammed with stuff - tip boxes, pipettes, trash etc. This all casts shadows which will leave wide areas untreated. Then UV light will only be active on the outside of tubes - so if the inside is contaminated, it will not help. It generates a false feeling of safety on the other hand.
4. Keeping pipettes clean on the outside is important here.
prevention is a good start...
1) separate work areas with separate equipment for preAMP and postAMP (separate pipetters, separate pipette tips, separate reagents (ddH20, etc)
2) aliquot new reagents (i.e. primers etc) so that if there is a question of contamination you don't loose the whole lot
3) Clean work areas and pipettes regularly. We use a bleach solution and we also use blotter paper that is discarded frequently
4) for some things we use filter tipped pipettors to minimize aerosol.
5) make master mix and aliquot it to individual tubes / wells. Then add DNA.
- Badges
- Science method