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Generating a cell line using sleeping beauty transposase and a gene that contain

Cloning SfiI Sleeping beauty

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#1 derkuli

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Posted 13 September 2020 - 09:20 AM

I need to generate multiple stable cell lines and am intrigued by the sleeping beauty system in particular a series of constructs that have been made available on addgene which have a lot of options for selection markers and fluorescent tagging (Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Kowarz E, Loescher D, Marschalek R. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821)

The plasmid contains flanking SfiI restriction sites for cloning the gene of interest however one of my genes contains the sequence, ie GGCCNNNNNGGCC.

I?d greatly appreciate help figuring out the best way to handle this. My current thoughts are that I could 1) make a substitution in the gene sequence so that I?m coding for the same amino acid but no longer have the recognition sequence, 2) create my forward and reverse primers as I normally would to generate the correct overhangs after SfiI and then just run the digestion and separate on a gel and cut out the band that would correspond to the digested DNA with the full gene intact since as I understand it SfiI shouldn?t cut at all three sites Since it needs two sites a strand of DNA to cut (i?m not 100% sure as I found this paper which seems if you let the digestion continue to run it?ll eventually cut each site but this was circular DNA so I?m not sure how that would translate: J. Mol. Biol. (2004) 339, 53?66. My other concern is the difference in weights of undigested DNA and my desired product and my ability to separate them.

I would greatly appreciate any help or suggestions.

#2 OldCloner

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Posted 02 October 2020 - 11:55 AM

Hi derkuli,

 

I have worked with SfiI, and was able to cut with it even though I had only a single site in my plasmid.  Apparently, the enzyme can bind to another site in another plasmid molecule- the two sites don’t need to be in the same sequence, although that speeds things up.  Here is a good source of information about enzymes that need to bind to multiple sites:

 

https://www.neb.com/...ti-site-enzymes

 

So you might need to use your strategy #1 to eliminate the SfiI site in your cloned CDS without changing the resulting aa sequence.  If you are concerned about having to defend this alteration in a publication, you can always do an extra step and mutate the internal sequence back to the wild-type SfiI.  Or you can try a partial digest with SfiI and hope that the two flanking SfiI sites get cut preferentially to the internal one, and that you get enough of the full length insert to purify on a gel. There’s no guarantee the internal site won’t get cut preferentially or as often as the flanking sites.  Enzyme kinetics can be influenced by the intervening sequence (the NNNNN part) in a split recognition site, so who knows?

 

If I understood your “other concern” at the end, you want to know how to tell whether you have managed to cut restriction sites at the very ends of your PCR product, since the cut fragment will only be a little shorter than the uncut product.  Well, I used to use proofreading polymerases in my PCR, then use a BLUNT-TOPO cloning kit to clone it into the TOPO vector without cutting it. Then I’d sequence to make sure there were no PCR-induced errors. THEN I’d cut the insert out with the enzyme for the sites I had added into my PCR primers, and gel purify the insert. With this extra step I never had any doubts about whether my sites got cut or not.  Anyway, some enzymes don’t like to cut near the ends of linear fragments, and this solves that problem, too.

Hope this helps. Good luck.







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