Amplification in water control but not in samples
Posted 14 August 2011 - 08:44 PM
Posted 15 August 2011 - 07:05 AM
So you should see no results at all in that water control sample.
Are you sure you are not mixing up something here?
And its possible that not all 7 samples are transfected like they should.
Altough: if it worked before.. and now it doesnt.. something is wrong (but you could have figured that out allready if you see a negative control that forms a band)
Edited by pito, 15 August 2011 - 07:06 AM.
If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.
Posted 15 August 2011 - 08:38 AM
1. alicuote and prepare your own working materials because when many peoples use the same reagent there is a chance that somebody contaminates it and then the rest of user will have trouble with their reactions. I always prepare a box for ea. student.
2. prepare the area that you are using for the pcr (decontaminate and place all materials that you will need) so you avoid to stand up to looking for something in the middle of work, thats a way of introducing contaminants to the area and in the reaction. Also is good idea to have a pipet set for pcr use only and when budget allows it use filter tips.
3.Do a master mix of the reaction (all except template), adding a reagent to each tubes rise the opportunity for a mistake and contamination. I always prepare an excel form with the reagent, quantity in each tube and how much I need for Xsamples (plus 1).
4. When placing the tubes on the rack make sure no one falls (if falls throw it away the tubes cost a few cents reactions that fails cost more money).
5. Pipet the master mix to all tubes (if you do it with care you will need 1 or 2 tips only), then add the template (one tip per sample), close the tubes after adding the template.
Hope this help
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Posted 15 August 2011 - 11:18 PM
Just to clarify, I meant that these PCR conditions and primers had worked previously, not that my specific samples had been shown to have Neo before, so it is entirely possible that only one of them has it (although somewhat unlikely as it was selected for).
Our lab has a seperate area for PCR with filter tips and I did make a master mix for it, but I realised that I had used MQ H20 from my bench as opposed to the PCR bench MQ H20, it's possible that that is where the contamination came from, although it doesn't explain it only being in only the water control.. I repeated the PCR and obtained a band for a single sample (the same one) and nothing in the water. Seems like it was just a matter of some contamination there and that my selection did not work.