same weird weird staph transformation results - any idea what is going on? (Nov/25/2009 )
I have some results!
Yesterday I decided to grow some RN4220 colonies from the Kan 600 ug/ml plate (this is the transformation plate, I picked some which appeared sooner than the others). I grew them in 3ml B2 overnight in 150 ug/ml Kan. Today I extracted the plasmid from them and run. Surprisingly that 3/5 of them show the plasmid-like band (as far as I know, RN4220 doesn't have native plasmid, right?). The 2 other lanes are very smear (I think it is just proteins, not plasmid). I checked them by PCR using the primers for the operon region on my plasmid (around 18Kb) and also use this plasmid as the control. You can see in the figure below, the control result is very good, but the 3 expected lanes showed something similar to the band I need. I don't know why these bands are so weak (I used 1/10 the DNA amount of the samples on the left).
I need to confirm my result by digestion, but I hope they're correct. So it means even we can't exclude the Kan resisted negative control, there are chances to get the correct colonies among them by choosing the "elite" colonies (if my result is correct, the main proportion of these colonies is positive).
How about you, plantonika? maybe you can succeed if try to screen like that ^^.
Hey Quasimondo, sorry for the delayed response, but I've been terribly busy and with no news in the transformation experiment. However, I did as you suggested and ran a PCR for some colonies regardless the negative control result.
I don't have the picture in this computer, but in general, I had no bands for the colonies in the negative control plate, and 3/5 from the transformation plate had bands, one had double bands and the other two only one.
Now I am digesting to see if I get the plasmid and the insert band. I will let you know as soon as I have the result. How was your confirmation digestion? did it work? I hope mine works.
Good luck and thanks for your imput!
planktonica on Dec 10 2009, 04:30 AM said:
I don't have the picture in this computer, but in general, I had no bands for the colonies in the negative control plate, and 3/5 from the transformation plate had bands, one had double bands and the other two only one.
Now I am digesting to see if I get the plasmid and the insert band. I will let you know as soon as I have the result. How was your confirmation digestion? did it work? I hope mine works.
Good luck and thanks for your imput!
I didn't perform the digestion because the plasmid is very large and maybe not very accurate (my control plasmid is from E. coli, the samples are from Staph, they might be different a little bit). Actually I transformed these plasmid back to E. coli and grew them on Amp plate (because this is the shuttle vector with AmpR gene for E. coli and KanR for Staph). I get some colonies and will extract plasmids again to compare. 90% that I succeeded ^^