I performed a PCR mediated deletion of my vector which was carrying an unwanted gene (Chey 183 bp). I designed two primers with desired restriction sites in order to clone any gene in place of Chey as the Chey gene was carrying no restriction site at it's 5' and 3' sites. Both the primers will run opposite to Chey gene excluding its amplification while producing intact plasmid backbone. Before doing such PCR I did simulation and got a 4516bp band excluding Chey gene. On agarose gel also I got a band of 4516 bp size. Now I am worried if I need to go for Dpn1 digestion or I can simply use the PCR purified modified plasmid for cloning?
Hi,
Have you extracted the amplified backbone from an agarose gel? If you were able to distinguish clearly between the linear and the circular form of the plasmid, you do not have to remove the PCR template by DpnI treatment. However, adding some DpnI is usually helpful.
However, you should remove the template for your initial PCR. If this plasmid remains in the reaction, it would compete with your newly constructed plasmid during transformation.
The DpnI digest only takes an hour and can usually be done in the same buffer that the PCR was performed in. It's quite simple and reduces WT contamination of your product vector.
Regardless of whether you do the DpnI digest or not, you should still transform and streak your product to ensure purity of your vector, verify the deletion, and possibly check the fidelity of your polymerase. This will identify a successful deletion anyway, but the DpnI digest increases your chances of identifying successful deletion first time.
Doing DpnI digestion of the PCR mix degrades your template DNA. It is very simple and useful for your subsequent steps.
Good suggestions above. Like Boas Pucker, I would use DpnI even on a gel purified band since it is easy to do and one band will usually contaminate the other, especially if the gel was not run slowly or if there wasn't a huge separation between bands. I also would think about what Adrian P Campbell asked about the fidelity of your poymerase--which one did you use? Was it a high fidelity pol? If not, I would repeat the PCR.
Lastly, the Chey gene may not have had convenient 5' or 3' restriction sites for use to mediate deletion, but it might contain a unique site. As such, before transformation, you can digest your plasmid or mix with that enzyme and the majority of the background DNA that contains that site will linearize and will not produce background colonies. If you have DpnI, however, then you have multiple sites on the original plasmid to at least linearize it.
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