how to check the insertion of a gene fragment into plasmid - (Jun/29/2008 )
Hi all
i've inserted a gene fragment (1428 bp) into EGFPC1 plasmid but i can't digest it with restriction enzymes to check its entering while the fragment insert check with related primers has responded so we are sure about its entering into plasmid. moreover when i run the inserted plasmid on the electrophoretic gel it shows a higher weight than been expected
Thanks in advance
sorayya
i've inserted a gene fragment (1428 bp) into EGFPC1 plasmid but i can't digest it with restriction enzymes to check its entering while the fragment insert check with related primers has responded so we are sure about its entering into plasmid. moreover when i run the inserted plasmid on the electrophoretic gel it shows a higher weight than been expected
Thanks in advance
sorayya
If you have cloned it through restriction digestion u should able to release the insert with restriction digestion.
If it is translational fusion u should be careful with the reading frame.
u can confirm it by sequecning .....
Hi sorayya,
sometimes after the inserted a gene into a plasmid, we didn't cut the same restriction enzymes to check it although the PCR is OK. please, check your gene sequence has any restriction area by NEB Cutter 2.0 (web: http://tools.neb.com/NEBcutter2/index.php). then, cut the new 2 restriction enzymes or 3, etc., in new options to check it. it don't need the same enzymes to do it. if your PCR results is ok, i think two results: firstly, your gene is in there, and secondly, your gene is in ther but twice. you should find new restriction sites in your gene or the recombinant plasmid.