DNA cleanup after linearization, before transformation? - (Apr/08/2014 )
Hi all,
I'm curious: most protocols I see on transforming Pichia pastoris (which integrates a linear vector), calls for cleanup after linearization.
Basically, this is how the protocol goes:
- Linearize vector which contains gene of interest
- Cleanup linearization reaction, which apparently removes the salt and the restriction endonuclease used in the linearization process
- Combine the cleaned-up, linearized vector w/ electrocompetent yeast, and transform
- The linearized vector will be integrated into the genome, and this is how expression happens.
My question is: can I eliminate step 2? i.e. will leaving the salt and the enzyme in the transformation reaction ruin the transformation?
Thanks!
--PRU
Hi all,
I'm curious: most protocols I see on transforming Pichia pastoris (which integrates a linear vector), calls for cleanup after linearization.
Basically, this is how the protocol goes:
- Linearize vector which contains gene of interest
- Cleanup linearization reaction, which apparently removes the salt and the restriction endonuclease used in the linearization process
- Combine the cleaned-up, linearized vector w/ electrocompetent yeast, and transform
- The linearized vector will be integrated into the genome, and this is how expression happens.
My question is: can I eliminate step 2? i.e. will leaving the salt and the enzyme in the transformation reaction ruin the transformation?
Thanks!
--PRU
No need to double post - bob.
The clean-up is not really necessary, however sometimes a high salt concentration in the digestion mix ruins the posterior electroporation. In this case you can:
-perform the clean-up or
-carry out the electroporation with less volume of DNA mix
It is also easy to do a drop dialysis of the digestion, which will remove the salt. Millipore VWSP 0025 membranes floated shiny side up on pure water in a petri dish. Pipet the diestion on top of the membrane, let it sit for 30 minutes, pipet it off.