We would like to put a 12-20 aminoacid linker into a recombinant protein expressed in E coli. We've had good experience with Quickchange-like protocols; but never tried with 36-60 nucleotide insertions. Is Quickchange (PCR-DpnI) likely to work? Complementary or offset primers? Or, would you recommend conventional cut and paste instead? Many thanks in advance!
HI Andrey,
in my research carrer I alwys used overlap extension PCR to add such modifications to my sequence. I agree this is more tedious, but works quite well and in my hands it was much more robust and eays to perform than Qucikchange modification. For example I have added a T2A site by using overlap extension as there were no restriction sites present. Let me know if you need more details
Sven
Quikchange should be able to handle these large insertions. The Quikchange primer design tool (https://www.chem.agilent.com/store/primerDesignProgram.jsp) really helps to get the primers that you need right.
The challenge that you will have here is primer length. To insert 20 amino acids you will need to make really long primers - probably 100 bases. You'll almost certainly need these to be HPLC purified as even the best primer synthesis companies only get a 99.5% incorporation rate, and over 100 bases this will give you a substantial proportion of primers with a base missing if you don't purify. Primers with a base missing massively affect the success rate of Quikchange.
Once you get to that stage, you might be forgiven for thinking "wouldn't it be quicker, easier, and even cheaper (even before you factor in your time) to get a gene synthesis company to make it for me?". That depends on the length of your protein and how many inserts you want to make. It's a question that is always worth asking nowadays.
Good luck!
Dear Andrey
i never use quickchange for long insertion..but i'm using PIPE cloning which is quite similar and with this I was able to insert and/or replace several signal peptides (LENGHT 15-20 AA) required for the secretion in mammalian cells and in the most cases (80-90%) it works very well.
Sometimes you can obtain some strange rearrangments but in the most of cases this problem could ne solced just sequencing more colonies.
you can find more details about PIPE cloning on my blog https://proteocool.blogspot.com/ at PAGE1
good luck
Manuele
Manuele, Nicholas, and Sven --
many thanks for the answers! After having read your suggestions, it appears that the primer length might, indeed, be problematic, and getting high-purity long oligos might take a while (e.g. IDT europe may take 1-2 weeks.... rediculous, b.c. in the late 90s when labs had their own synthesizers, this would be an ovenright synthesis, +2 hrs of HPLC :) ). Thus, my colleague has suggested an add-on PCR of the entire plasmid using two non-overlapping primers (each one having 1/2 of the intended linker, i.e. shorter and not needeing purificaiton). One then uses a polymerase that does not add A's at the end. Then one phosphorylates the linear product and self-ligates.
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