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Insert 30nt at C-terminal

insertion

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#1 Biogareth

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Posted 23 January 2014 - 01:16 PM

Dear all,

 

I am going to add 6histag in C-terminal of my target protein. It is my first time to insert such big pieces in a gene. I wonder which protocol could be useful for this purpose? I have used QuikChange II XL Site-Directed Mutagenesis before to add 6 nts. Will it work in this case?

In addition, I was told several aminoacids should be added between his-tag and target protein. Normally how many and which kinds of aminoacids should I add into? Are there other points I have to keep in mind for such insertion? Many thanks in advance!

 

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#2 phage434

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Posted 23 January 2014 - 02:25 PM

I think the easy way of doing this is to design two primers for amplifying your entire plasmid minus the stop codon on your gene. Then add the bases for your his tag, a rare restriction enzyme (such as NotI), and some extra bases that can be cut off. On the other primer, add a similar NotI cut site and some extra bases. PCR, purify, and then cut with DpnI and with NotI. Heat kill, ligate, transform.

 

For a tag, you could try something like GGGSGGGSHHHHHH-stop

Some people like to add 1-2 more his codons.

 

Of course you need to verify that your plasmid has no NotI (or other) sites on it.







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