Hi all. I want to insert an enterokinase site in my plasmid using PCR mediated site directed mutagenesis, so is thier any standard protocol for this. My insert size is 14nt and plasmid 4.2kb
Hi there,
There are two main approaches : the one from Quickchange kit from Agilent(https://www.agilent.com/cs/library/usermanuals/Public/200523.pdf) where the sequence to be inserted lies right in the middle of both complementary primers. There is a variant protocol where insertion lies only on 1 primer ( this paper should help to figure out the strategy: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629768/).
The other approach is the one from NEB Q5 mutagenesis kit (https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit) where insertion sequence is at the 5' of each primer.
A You can try Polymerase Incomplete Primer Extension method where you have to just design primers with the bases you want to add and just do one round of PCR and do transformation to get desired insert in your plasmid. With this method you can delete any number of bases from your insert. Please find attached paper which will help you to design your experiment. I have added TEV protease site at N-terminal of my protein which was 21 base pair successfully. Hope this will help you.
Sounds awkward and expensive for such a short construct. I think what you really want to do is order a synthetic gene containing the desired modification.
If your plasmid has a phage replication origin (like many common plasmids) you could use Kunkel mutagenesis. I can provide the detailed protocol. First you have to obtain single strand DNA packaged into phages. Once this is done, the technique is extremely simple- an overnight reaction and a transformation.
But perhaps its simpler for you to perform quickchange mutagenesis on your double-strand plasmid. You dont need any kit, you can reproduce the same protocol of the quickchange kits (available elsewhere) with your own reagents. Try to use a high fidelity DNA polymerase. The protocol consist of 1. a single PCR reaction, 2. digestion of PCR products with DpnI, 3. transformation of competent cells with digested PCR products. Thats all.
I use very simple methods which I have though myself and it works pretty well. You just need two primers, one should be phosphorylated (or you can phosphorylate the primer in the lab using PNK enzyme), the second primer will have the desired mutation at the 5 prime end. Please design primers from the region where you want to make the site directed mutagenesis. One primer will go in the forward direction and another will be in the reverse direction. There will be no overlap between the primers. and one primer will have the insert seq as over hand.
Use these primers in a PCR reaction by an high fidelity enzyme. I use hot start fusion from thermo. Amplify the par reaction 25 cycles. After the PCR do the DPN treatment to chop off the native plasmid DNA. Please check at this step whether you can do the don treatment in your per reaction directly or you need some supplement. After DPN, take some portion of PCR directly and set up a ligation reaction. transform the ligation product. Most of the colonies will have your insert.
cheers!
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