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how to switch to a gene in a vector using PCR or restriction enzymes? - gene is in pIVS2 vector (Aug/10/2007 )

I got a construct from a lab with human gene 1, i want to replace this gene with another human gene 2 without changing anything in the vector. This is a transgene construct.

Please let me know if my strategy is good enough

1. Design two primers (30+ long), one before the start of CDS of gene 1 (forward) and second starts after the stop codon (reverse primer) of gene 1.
2. Do the PCR-------------(that will remove the 520 bp long gene 1 )
3. I want to have 6-7 His tag right upstream of my CDS (to differentiate from the background level of mouse gene product), so shall I design 2 primers with restriction sites (not present in vector and gene 2), then do the PCR of the vector to incoporate those rest sites.
4. Then do PCR of gene 2 with primers having the same two rest sites.
5. Use these rest enz to digest vector and and the insert.
6. ligate them

I would appreciate any suggestion/comments.


the PCR of vector seems good to me.
I would recommend to do the pcr on digested templates


QUOTE (fred_33 @ Aug 10 2007, 11:04 AM)
the PCR of vector seems good to me.

********* u mean to say the PCr design to remove 520 bp long CDS is fine??*****

I would recommend to do the pcr on digested templates

I do not understand this, sorry, can u pls explain?



Find sites in vector which you could use and not present in gene2. look for the same sites used to clone gene1 provided its not present in gene2.

Design primers for gene 2 with restriction sites (not present in gene 2). Both upstream and downstream.

Design a second 5'primer to add the His tag.

Leave enough bases on the ends of the primer after the restriction sites for adequate digestion.

Amplify gene2, digest vector and PCR with the respective enzymes.

Ligate vector and PCR products.

I hope I haven't missed out anything.