Hello everyone,
This is my first time working with plasmids. My main goal is the expression and purification of proteins of interest. I am using a plasmid with CDS of another protein, which I need to replace with CDS of my protein of interest.
So now I have linearized the plasmid by using XbaI. On the other hand, I have made primers for the template that I want to insert by using vector sites where I want to insert it. but I am still confused about the protocol related to recombination and DpnI. Your advice and suggestions are highly appreciated.
DpnI degrades methylated DNA. This is to destroy the parental plasmid so that only your newly synthesized recombinant plasmids will remain and are able to be transfected. Without DpnI treatment there is a possibility that some of the parental plasmids will recircularize by blunt end ligation giving rise to colonies that are false positives.
I assume you are using PCR to carry out your recombination and not Lambda red recombineering?
Thank you.
Yes, I am doing this for recombination, and after constructing the plasmid, I will use BL 21 expression system. I am not doing Lamda Red.
So, after linearizing my plasmid, I will do the DpnI step and do the ligation of my template?
The second question is I have inserted the restriction sites in the primers that I will use to amplify the template. Is this enough to make sure that the template will be ligated to the linearized plasmid?
Are you just doing a restriction digest and ligation, in which case recombination is not the right term, or are you doing a round of PCR amplification to extend the ends of your insert to include the plasmid backbone? Maybe you could be more specific about what you mean by "vector sites"? Do your primers have homology with the ends of the linearized plasmid?
If you are doing a restriction digest followed by ligation you only need the DpnI to destroy the template in your PCR reaction. Applying it to your linearized vector must be avoided!
I assume the restriction sites in your primers are also XbaI? In that case you need to also do a restriction digest followed by a PCR cleanup on your insert before ligation. As XbaI is not a blunt end cutting enzyme, performing a blunt end ligation of your insert would have a very low yield.
After linearizing the plasmid with XbaI, you should first proceed with the ligation of the PCR-amplified insert (which contains the CDS of interest and has been similarly digested with XbaI) into the plasmid. Post-ligation, treating the mixture with DpnI is crucial to digest the original, non-recombinant plasmid DNA, which is methylated and thus a target for DpnI. This step enriches for the newly formed recombinant plasmids. Following DpnI treatment, you can proceed with the transformation into BL21 E. coli cells, which is an efficient system for protein expression but does not involve Lambda Red recombination. The subsequent steps will involve selecting and confirming successful recombinants before moving on to protein expression.
Do the DpnI digest after PCR amplification and before the PCR cleanup. Do not do it to your destination plasmid.