I have inserted a gene approximately 2000bp for protein expression in pCS2+MT vector. But now I need to delete 200bp from that gene. If I delete it. it will remain inframe.
Whole plasmid PCR of-course. To religate your PCR-amplified plasmid, either introduce a suitable restriction site in your primers or use phosphorylated primers and go for blunt end ligation.
Hi,
Using PCR to amplify the whole plasmid followed by gibson assemblly can easily make it.
Good luck!
Whole plasmid PCR of-course. To religate your PCR-amplified plasmid, either introduce a suitable restriction site in your primers or use phosphorylated primers and go for blunt end ligation.
Simplest option is, restriction enzymes in that 200 bp which you want to delete. Select such restriction enzyme/ ezymes that do remove this fragment and go for ligation.
Hi,
Falseplanet Jia and Syed Ali's suggestion is probably the easiest way to go. You could also look at the Quickchange mutagenesis kits, but I've no personal experience of doing such a large deletion with them.
To answer your other question, if the deletion is exactly 200bp then the sequence downstream from it will be out of frame and so not translated properly (200/3=66.66). The deletion needs to be exactly divisible by 3 to keep the frame right (e.g. 201/3=67).
The best thing is to check the translation using a DNA handling program, or an online translation resource site before generating primers and doing the experiments.
-Richard
Hi, i agree with Richard. But it is not clear whether 200bp lies in frame or outside it. If it is outside than no difference.
Dear Mr Ali,
Yes I am trying blunt end ligation but the primers not phosphorylated. I am am treating by kinase after PCR than T4DNA ligase but no clones are found.
Dear Richard, Yes you are right, the deleting portion exactly 120 bp so it will be inframe again. 120 bp is an exone.
Thanks
I'm a little confused here. Are you wishing to delete an exon from between two introns, or is it the sequence for that exon in the cDNA?
Hi
Finally I have got it by repeating in the 2nd time. In 2nd time I did not purify the PCR product by using agarose gel. PCR product was directly used for kinase and ligase treatment.
I confirmed approximately 500bp around the deleted portion but still worried about the backbone for any changes.
Thanks for your advices
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