5 replies to this topic

[ironman88]

Hi,
I am trying to introduce a MCS of about 4 RE into a region without any known restriction sites. any suggestions on how to achieve this cloning?

thanks.
ironman

[rkay447]

Use the Stratagene QuikChange mutagenesis protocol.  Design primers that contain the desired sequences and use them to amplify the rest of the vector.

[phage434]

It would probably be easier to do a normal PCR with overhangs encoding the desired MCS sites (two on one, three on the other) along with 4-6 bp of overhang.  Then, column cleanup, cut with the 5' restriction enzyme and DpnI, religate, and transform.

[christy]

u can try with linker. or clone first gene in the available site, during that amplification include restriction sites in the forward primer or reverse primer. only thing is ur primer will be bit lengthy. that doesnt matter. I did that. u can include n number of sites.

ironman88, on 24 November 2010 - 09:11 AM, said:

Hi,
I am trying to introduce a MCS of about 4 RE into a region without any known restriction sites. any suggestions on how to achieve this cloning?

thanks.
ironman

[ironman88]

thank you for all your comments.

how do i cut my parental sequence in the first place to put in the linker or clone in the gene? there are no restriction sites on the vector region that i am interested in.

my vector is 7-8kb. i am worried that with site directed mutagenesis pcr of the whole length, i will also introduce mutations. are my worries unfounded?

maybe i don't understand what your suggestions are.

thanks for clarifying and helping.
d

christy, on 24 November 2010 - 04:33 PM, said:

u can try with linker. or clone first gene in the available site, during that amplification include restriction sites in the forward primer or reverse primer. only thing is ur primer will be bit lengthy. that doesnt matter. I did that. u can include n number of sites.

ironman88, on 24 November 2010 - 09:11 AM, said:

Hi,
I am trying to introduce a MCS of about 4 RE into a region without any known restriction sites. any suggestions on how to achieve this cloning?

thanks.
ironman

[phage434]

You don't have to cut the parent plasmid -- the PCR will bind to the primed locations on the circular DNA and create a linear fragment, with the 5' overhang bases on each end (which incorporate your MCS restriction sites).  When you cut with the 5' enzyme and ligate, you will recircularize the linear fragment creating a plasmid you can transform.

You probably don't much care about mutations in the plasmid.  Ones that replicate and have the correct resistance (i.e. the ones that successfully transform) are probably what you want, and any other changes are probably OK (except perhaps for introduction of more copies of a restriction site you want to use).