Re: [DIYbio] Re: stable transformation of chlorella vulgaris

Inverse PCR is also going to be difficult unless you have some sort of mechanical DNA shearing method. Common enzymatic and chemical shearing will break the DNA down into pieces that are just too small.

I would advise using a splinkerette pcr, or following the protocol that I've attached. Both are simple, rather elegant ways to ensure your PCR products come from the desired location in your genome. 

-SG 

On Thu, Jul 12, 2018 at 9:31 AM, Scott <synbiofablab@gmail.com> wrote:

You most definitely don't want to do PCR on genomic DNA with random primers! Inverse PCR, as I mentioned earlier, is the way to go. It has been use in all sorts of species from bacteria to mammalian cells. You can even do plasmid rescue if your transgene contains a bacterial origin and selection marker but inverse PCR is the way most people look for integration sites. There is a variant where one ligates a primer-bearing adaptor to the digested genomic DNA followed by PCR with a transgene-based primer. You can get kits for that for the young folks who are flush with money and don't like getting their lab coats dirty!

Cheers,

Scott

https://opensciencenet.org/

On Thursday, July 12, 2018 at 12:02:25 AM UTC-7, Ravasz wrote:

...

Finding the genomic integration site will be another problem, but this might be done with a simple PCR and sanger sequencing of the product, using a primer within the plasmid and a bunch of random primers. This may or may not work, if it doesn't then probably some high throughput sequencing will need to be done, which will quickly get rather expensive if you want to test multiple single-cell colonies

...

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