I want to perform site directed mutagenesis to a sequence I have cloned into a pGL4.1 vector, but I'm not sure which vector to clone the mutants in once mutagenesis is validated. I want to clone into LNCaP cells (prostate cancer cells) and perform ChIP and qPCR analysis for target genes. I am a first year graduate student and not sure how to go about deciding.
Hi Ariel,
I don't know the answer to your question, but I'd like to try and help you in a different way. As a first year graduate student you are probably going through your rotations and you should be able to ask this question to your mentor or to more senior graduate students or postdocs in the lab; I would start asking any of these people, they are likely to know the project and should be in the position to help you out. If no one in the lab is able or willing to help you, I suggest you look for a different lab and make sure you find a friendly and collaborative environment.
Good luck!
It depends on what you will do with those cells.
If you are just going to transfect the cells with the mutant plasmids, make sure the plasmid you select has a promoter that will express the mutants once inside the cells.
If you're going to try to make RNA from the mutant genes in the plasmid then you will need a plasmid that has an RNA promoter in it as well as an SV40 poly A tail. For this reason we usually use pCS2+ as our plasmid for mutagenesis.
I totally agree with Carlo; check with your labmates before you proceed.
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