Problem with colony PCR - MBL detection in Pseudomonas aerugfinosa (Jan/02/2011 )
thank for your suggestions.
you have explained in good detail.
presently i am thinking of two possibilities for my inconsistent results. first is that my primers might have been mishandled. secondly problem with my template.
do u think DMSO might help me in better results?could you guide me in its concentration per 25ul reaction mixture?
I ve been sub-culturing the same isolate every day for the past one and a half months to use the fresh sub-culture everyday. May be you are right. I think one of the options can be if i do a fresh sub-culture from my preserved stock of the bacterial isolate.
Can we be sure about the amount of DNA in the diluted McFarland suspension e.g. by optical density determination?
I use DMSO in almost all of my PCR at 3-5% concentration. But my main organism is B. pseudomallei and not P. aeruginosa.
I use P. aeruginosa in some of my controlled test and I also had cloned some genes from it.
I would suggest you to purify the DNA (using column or kits or any reliable established methods) as a control template for your PCR. Optimize your PCR with the purified DNA. Once you had done this, try run your colony suspensions sample.
For my application, I do not require to know the exact amount of DNA. You can do rough estimations of the amount of bacteria by comparing with McFarland standard, for the colony suspension method you use, you did not extract the dna out from the cells, and I don't think is appropriate to measure the amount of DNA.
And, please check your culture whether your culture is pure. Do API20NE test to confirm identity and/or 16s. I used to be given B. cepacia isolates which was handled down for "generations", I tried my primers and it doesn't work out for many isolates, and my boss blame me for my bad technique. After numerous failures, I decided to check the identity by using 16s and I found that most of the B. cepacia isolates given to me was actually bacillus sp and my primers actually did not failed after all. So, please double or triple confirm your isolates before you start, I learn it the hard way.
Maybe you can share your applications or what you intend to do here, so we can discuss a little further.