I am working with plasmids containing aion channel, with the goal of eventually using them for transfection in Hek cells. The problem I am having is preparing these plasmids at the bacterial transformation stage. I have 2 different channels (both from he HCN family), on of them (HCN2) grew perfectly on the first try in XL1 Blue cells. However I am now doing point mutations on the channel (using a Quikchange kit) and I cannot get a colony that has my intact channel. Additionally I am trying to use HCN1, another member of the same family, and it is giving me similar problems to the mutation reaction. Here is what I have tried so far:
1. I am using internal channel specific primers to screen picked colonies for the presence of my plasmid. PCR of the unmated HCN2 plasmid produce a clean band of the appropriate size. PCR of the mutation reaction prior to transformation produces a single band of the right size. but PCR's of the picked colonies for the mutants do not, they show multiple bands.
2. Used Stbl2 competent cell to hopefully prevent recombination of the plasmid,but the pct's looked the same as the XL1-Blue.
3. Tried incubation at 37 and 30 degrees, and decreasing the antibiotic concentration, but still the same problem
I have tried these things with both the HCN2 mutation reaction and the wild type HCN1 plasmid and have had no luck.
Any advice would be much appreciated! Also, if there are any extra details that would help please let me know
Thanks!
Anna
What is happening? No colonies upon transformation, or the insert is disappearing? Or is the insert there, but no mutation?
Perhaps the mutations cause the protein to be toxic, although unlikely. Perhaps there is secondary structure in the plasmid affecting Quikchange. What plasmid are you using? Maybe you need to move your insert into a small cloning vector to avoid recombination issues.
Hi Anna,
Ion channels can be problematic.
From the information you have supplied it does sound like you are having a problem with your plasmid recombining. Some thoughts and ideas:
So do your genes have any sequences that are repeated or have high homology? This homology could make the recombination worse when you run the Quickchange mutagenesis, as you will be recombining two separate single strands. Any hairpins formed will not be visible in the PCR due to the denaturing temperatures of the reaction, but will be present when you transform the E.coli.
Have a look into generation of synthetic gene versions, as any unwanted sequence homology can be removed. I've had some success with improved stability of an ion channel gene this way.
Other methods for mutagenesis such as SOE-PCR might work better than quick change.
Are there any genes in the plasmid (resistance markers etc) that are in the same orientation as your gene. There could be message read through from these into your gene, even low level transcription of your gene might be toxic. If possible look into using a vector with all other genes / promoters in the antisense orientation.
-Richard
Your problem is not due to plasmid itself recombination, it is caused by low mutagenesis efficiency . Try to read the paper on http://www.biomedcentral.com/1472-6750/8/91 and repeat your mutagenesis follow my lab lab protocol as attached if you want.
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