homologous recombination in E coli - (Jul/05/2008 )
Hello everyone,
I am trying to do homologous recombination in DH5 alpha but has no success.. the homologous bits are about 20 bp? Is it enough?
Has anyone done it and suggest some advice?
Thanks 
the smallest fragment I havw done so far is 300bp....
How long is the ligation?
Recently I found that u can do at 25℃,5minutes and
it always works
No, it's not a ligation. It's homologous recombination.
Basically, u PCR a gene with bits of homology to the vector (~ 20~ 40 bp), then u transform both the cut vector and PCR product and hope that recombination occurs. This way, you dont need to cut the PCR product with restriction enzyme.
Have a look at the paper attached...
I tried, but no success.. just wonder if anyone has tried and could give some suggestions..
Basically, u PCR a gene with bits of homology to the vector (~ 20~ 40 bp), then u transform both the cut vector and PCR product and hope that recombination occurs. This way, you dont need to cut the PCR product with restriction enzyme.
Have a look at the paper attached...
I tried, but no success.. just wonder if anyone has tried and could give some suggestions..
Why keep homology to 20bp? It is always better the bigger are the homology arms. I would go with 40. I also suggest using NCI recombineering system (Copeland).
I am trying to do homologous recombination in DH5 alpha but has no success.. the homologous bits are about 20 bp? Is it enough?
Has anyone done it and suggest some advice?
Thanks
Can you be more specific??!! are you trying to integrate a bit of DNA into the DH5 alpha genome? or do you want to genetically manipulate the DH5 alpha genome?? DH5 alpha is a rec- strain and hence homologous recombination is not possible. are you doing plasmid based? Kindly elaborate so that I can help you...
I am trying to do homologous recombination in DH5 alpha but has no success.. the homologous bits are about 20 bp? Is it enough?
Has anyone done it and suggest some advice?
Thanks
Can you be more specific??!! are you trying to integrate a bit of DNA into the DH5 alpha genome? or do you want to genetically manipulate the DH5 alpha genome?? DH5 alpha is a rec- strain and hence homologous recombination is not possible. are you doing plasmid based? Kindly elaborate so that I can help you...
Can you Recombine your posts?
Thanks BioBills,
No, not integrating into the genome, just cloning of an insert into a vector without using restriction enzymes and ligation. I included a paper earlier in this topic. It described using DH5alpha to do recombination b/w a DNA fragment and a plasmid. I followed the protocol and didn't get any result. There are actually many papers describing homologous recombination cloning in E coli DH5 alpha. Even it's RecA-, it can still carry out recombination through a process still not well understood.
Since it's RecA independent, the homologous bits only need 20 to 40 bp, not 1000 bp as in RecA-dependent recombination.
Cheers
No, not integrating into the genome, just cloning of an insert into a vector without using restriction enzymes and ligation. I included a paper earlier in this topic. It described using DH5alpha to do recombination b/w a DNA fragment and a plasmid. I followed the protocol and didn't get any result. There are actually many papers describing homologous recombination cloning in E coli DH5 alpha. Even it's RecA-, it can still carry out recombination through a process still not well understood.
Since it's RecA independent, the homologous bits only need 20 to 40 bp, not 1000 bp as in RecA-dependent recombination.
Cheers
hey thanx for the info.
An alternate paper that is much more recent is this: [attachment=4994:Li_and_E...LIC_2007.pdf].
T4 DNA polymerase and RecA protein are easy to get hold of. We are trying the system at present, so I don't have any data or suggestions for you. Hopefully in the next couple of weeks though.