I had maxi prep for plasmid, and I have problem with PCR for recombination also PCR with primer on the insert gene .
SO I am wondering is there a possibility that the bacteria could introduce mutation in the transformed plasmid ?
less tha 0.01% chance to mutate a single bp in plasmid during transformation. Above all if I assume that mutation could be the reason for your problem so it may be on one bp primer can attach if there is 1-2 mismatch (except 3'). So try to optimize your PCR and check either primer is working or damaged.
less tha 0.01% chance to mutate a single bp in plasmid during transformation. Above all if I assume that mutation could be the reason for your problem so it may be on one bp primer can attach if there is 1-2 mismatch (except 3'). So try to optimize your PCR and check either primer is working or damaged.
If you used a single clone from a re-transformation for the MAXI-culture, it is possible that this particular clone harbours a mutation which interferes with your PCR (as Nishat pointed out). To circumvent this, I usually use a small part of the whole transformation reaction for inoculation in midi or maxi prep.
But think also of other possibilites: Try analytic digest (in parallel to original plasmid) to confirm you have the right plasmid. Look at the quality of your plasmid prep (supercoiled band should be >50%). Residual ethanol from the prep can also be a problem for follow up reaction.
Good luck
Best
Christian
Please consider all the above suggestions. One important aspect not mentioned is the 'stability' issues of some plasmids. You have to use special transformation host, lower the incubation temperature (30C, instead of 37C), and 'slow' down the growth of 'unstable plasmids bearing mutational hot spots' to minimize the recombination events (DNA rearrangement problems).
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