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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

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.





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