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DNA cleanup after linearization, before transformation?

Transformation Pichia pastoris DNA Linearization

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3 replies to this topic

#1 Proteins-R-Us

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Posted 08 April 2014 - 05:48 PM

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: 

  1. Linearize vector which contains gene of interest
  2. Cleanup linearization reaction, which apparently removes the salt and the restriction endonuclease used in the linearization process
  3. Combine the cleaned-up, linearized vector w/ electrocompetent yeast, and transform
  4. 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



#2 Proteins-R-Us

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Posted 08 April 2014 - 05:50 PM

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: 

  1. Linearize vector which contains gene of interest
  2. Cleanup linearization reaction, which apparently removes the salt and the restriction endonuclease used in the linearization process
  3. Combine the cleaned-up, linearized vector w/ electrocompetent yeast, and transform
  4. 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.



#3 Binomial

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Posted 08 July 2014 - 02:29 AM

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 



#4 phage434

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Posted 08 July 2014 - 05:52 AM

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.







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