3 replies to this topic

[It'sNotMe]

Hi everyone,

I have a simple question here :

1. Is it fine to clone a glycosilated peptide's cDNA in an E.coli system? I understand that glycosilation is a eukaryotic post-translation modification and cDNA sequence should remain intact but does the cloning of cDNA in E.coli effects its expression in a eukaryotic expression system?

Thank you,
It's Me

[bob1]

It is fine, most cloning is done in plasmids that are propagated in bacteria.

[It'sNotMe]

Hi,

Thank you for your kind answer. I have one more question:

If I have a PCR product that I want to express in eukaryotic expression plasmid, pEGFP-N1. Is it fine if I inserted the PCR product into the pEGFP-N1 and propagate it in Escherichia coli (DH5alpha) and isolate the plasmid and transfect into HeLa cell for expression?

or

do I have to clone the PCR product into a cloning vector and the excised ligate into pEGFP-N1 for protein expression?

Sorry for the stupid questions, I am new to this..

Thank you,
It'sMe

[bob1]

It should be fine to do the first scenario.