I was doing mutation using PCR, the amount of plasmid that I took was greater than amount given in protocol for digestion of the parental DNA, incubated for more than 4 hours at 37C, then transformed it and plate was at 37C for about 18 hours and I got lots of colonies.
Can anyone tell what might have happened?
Um, well... it means you got lots of colonies!
How many do you mean by a lot? What controls did you include? How many did they give? Did you add antibiotic to your plates?
But really those questions mean nothing... the important one is have you sequenced any of your plasmid yet to see if you got mutants? Because if you did, then it doesn't really matter how many you got, as long as you have one with the correct sequence you can use!
If you don't get mutants, go back and follow the protocol!
Hi Vinita
I also agree with Richard above that...very less information is provided by you on above-mentioned experiments to point out any problems....with it....Provide some details of the experiments.to draw some conclusions.....However if you are getting a large number of clones (I assume a lot more than your previous experiments)..then there are chances of contamination per se or due to in sufficient antibiotics...
bye
Hi, there could be many possibilities, have u confirmed your pcr product on agarose gel. if ur pcr product is in high concentration then u would have large no. of colonies on plate. Since u have digested the parental DNA and only pcr product containing mutation will b present therefore there would b less probability of wt gene colonies. have u inserted any extra restriction site at the point of mutation to screen the +ve clones if yes just isolates plasmid DNA for at least 10 colonies do digestion to check +ve clone. if u dont have inserted any restriction site at the point of mutation then i would suggest u to go for at least eight colonies for sequencing to confirmed the mutant.
Well as all others said, how much is exactly a lot for you? and what about your negative control says? if the negative control gave only a few and your test plate has more than 10times the negative plate, then isolate the DNA and go for sequencing to find the right clone with the mutation!
Good luck.
Thanks all of you guys. Actually I was doing PCR for the first time and it didn't worked, which I get to know after DNA gel electrophoresis b'coz I didn't got the probable band. I am repeating it again.
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