Different sizes of PCR products from different competent cells - (Dec/11/2006 )
Hi!
Is it possible that the PCR products of the same construct from different competent cells have different sizes?
I have a construct and I transformed it into two different strains of competent cells, then extract the plasmid from these two cells. But when I PCR with the same primers, the product is with different sizes!! Is this normal or if anyone knows that if there's any problems??
PLEASE HELP ME~~
Thanks!!
That may happen if one of your strains has attacked your insert. Some strains will recognise insert DNA as foreign and begin to attack it with endonucleases. This leads to part of the insert being digested away. This is particularly problematic with larger inserts. There are strains available (genotype end- i think, end for endonuclease) that lack this endonuclease activity and are more likely to prevent the attack of your insert.
I never heard about it.
My be I don’t understand very well. 
killerkoz17:
I would think most of the commercial strains we use by rutine lack this this endonuclease activity.
Could you give some examples of one and other strains???
My be I don’t understand very well.
killerkoz17:
I would think most of the commercial strains we use by rutine lack this this endonuclease activity.
Could you give some examples of one and other strains???
Actually no. Not all commercial strains have their endonuclease and recombinase activity knocked out. As one would expect the more genes you knock out the sicker/slower growing will the strain become. The endonuclease and recombinase genes are part of the cell's normal DNA repair machinery.
You can break "commercial strains" into two groups.. cloning strains and expression strains.
Cloning strains have their endonuclease genes knocked out (how many genes will depend on the strain.)
Expression strains, generally have their endonucleases active.. because you want a health cell, with good protein making abilities for your expression.
Now cloning strains can be further broken down to those with their recombination proteins knocked out and those who are not. And there is a variation with some strain having more recombinations proteins knocked out. In some situations, people want the recombination proteins working, so large scale DNA manipulations can be conducted. Other times, you want to synthesis tandem repeats Mb long, or maxiprep very large plasmids, which require a very stable e coli strain.
So there is a large variation, in commercial strains. My feeling is that Abey303 has accidentally placed her plasmid into an expression strain and perhaps comparing it to another transformation which went into a cloning strain.
I think I knew that, but you put it in a very simple and clear manner!!! 
Now, it’s more clear for me!! 
Thanks! 