PCR contamination - (Aug/29/2010 )
sorry so my post was then not much help for your problem, sorry for this.
It looks as if you got a contamination in your chemicals, have you tried to change them and are you sure that nobody else used your chemicals?
And second: autoclaving does not necessary destroy DNA contamination on you pipetts etc., you should try and clean them with bleach, DNA-off or something similar. Do you have some of purchased ultraclean certified DNA/RNA free water in your lab (often also comes as component of extraction purification kits)? Try to use this water for the negative control, if possible try to do your PCR with chemicals from another lab that does not work with mice using their equipment....
Last but most important:can you post your PCR protocol? If the annealing temp is too low you might get unspecific priming with some DNA contaminiation. If possible run a gradient PCR with water only to see at which temp the band is disappearing. If the times are too long there might also be a problem with specificity.
Your primer stock could be contaminated. For PCR primers, I usually keep a concentrated stock and a more dilute working stock. Do you have a more concentrated stock that you can make a new working stock from? This would be a quick test
gebirgsziege on Mon Aug 30 13:40:40 2010 said:
sorry so my post was then not much help for your problem, sorry for this.
It looks as if you got a contamination in your chemicals, have you tried to change them and are you sure that nobody else used your chemicals?
And second: autoclaving does not necessary destroy DNA contamination on you pipetts etc., you should try and clean them with bleach, DNA-off or something similar. Do you have some of purchased ultraclean certified DNA/RNA free water in your lab (often also comes as component of extraction purification kits)? Try to use this water for the negative control, if possible try to do your PCR with chemicals from another lab that does not work with mice using their equipment....
Last but most important:can you post your PCR protocol? If the annealing temp is too low you might get unspecific priming with some DNA contaminiation. If possible run a gradient PCR with water only to see at which temp the band is disappearing. If the times are too long there might also be a problem with specificity.
gebirgsziege,Thank you very much! Only me use these buffers. and I also use UV to bleach the pipetts for 30min
my PCR protocol as follow
1.95℃ 2min
2.95℃ 30s
3.55℃ 30s
4.72℃ 30s
repeat step 2-4 35 times
5.72℃ 10min
Kaioshin on Mon Aug 30 13:42:08 2010 said:
Your primer stock could be contaminated. For PCR primers, I usually keep a concentrated stock and a more dilute working stock. Do you have a more concentrated stock that you can make a new working stock from? This would be a quick test
Dear Kaioshin,
I order the new primer and use the new one to run the PCR. but the ghost band is still there.
55°C is not too high, although not too low either. If possible try a gradinent PCR, or if you cannot, try 60 °C with a real sample and the contaminated water....
edit: try to clean the pipett in the inside as well!
gebirgsziege on Mon Aug 30 13:57:30 2010 said:
55°C is not too high, although not too low either. If possible try a gradinent PCR, or if you cannot, try 60 °C with a real sample and the contaminated water....
edit: try to clean the pipett in the inside as well!
Dear gebirgsziege,
how to clean the pipett in the inside. I use 75% alcohol to clean the pipett. Is it ok? Thanks again!
your lab manager should have a manual for this; otherwise contact the manufacturers homepage how to open and clean the pipett. Then use DNA off or diluted bleach to rise the parts that can be rinsed (see manual), let them dry overnight and re-assemble the pipett.
If you are using filter tips for everything, then you shouldn´t need to open the pipette (although its always nice to have clean gear!).
What did you prepare the new primer stock in? If you can buy some sterilised water and use that for making you stocks (and working soln), it might help.
What state is your autoclave in? Maybe you tips are coming out dirtier than when they went in? Try a new box of tips...
just my 0.02
Are you sure your primers can't amplify human DNA? have you tried to sequence your band?
second : have you tried cleaning the area you are working in, the PCR rack , .... etc with bleach 10 % ?
wishing you all the best