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Transformation of PCR product to get resistance with no plasmid
Very new in yeast field & needs an urgent help.
I need 2 introduce His3 resistance into yeast strain & want 2 know if it is possible to make transformation of PCR product to get resistance with no plasmid. If so what should the PCR primers include besides homology to the His3 gene in order to dig it out of genomich yeast DNA?
2. Can the PCR piece be than directly transformed into the wanted strain without the use of a plasmid?
3. If so what is the principle this transformation is working upon?
Thanks a lot
Last edited by leviuso; 07-14-2009 at 06:34 AM.
Re: Transformation of PCR product to get resistance with no plasmid
This is a very easy thing to do if I am understanding you correctly. Simply create primers to amplify His3 and then add about 50 bases on the 5' end of these primers that is homologous to the area in the genome you wish to put it. It will recombine via homologous recombination. Make sure to do a high efficiency transformation though, and I usually would plate this onto YPD then replica-plate onto selection even though its not an antibiotic.
I am not sure about what you are trying to do though, why would you want to put His back into the strain? And if this is what you want to do, you should probably try to put it back into the place where it was already deleted or mutated so that you can have its native promoter working for it, unless you will amplify with a promoter off a plasmid or something.
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