I have cloned my insert DNA into pBluescript SK+ vector, after this cloning I have performed site directed mutagenesis by using PCR then digested it by DpnI digestion. what control should I use for this to ensure DpnI complete diigetsion ? Can only pBluescript vector DNA be used as a control for DpnI digestion if yes so please explain how this control will ensure complete DpnI digestion of mutated clones?
Hello Monika,
You set the control at the same time when you run PCR for site directed mutagenesis. The control is the plasmid without the primer (the primers have mutations). You use the same amount of plasmid with the experimental samples to run the same protocol and treat DpnI. Then perform transfomation. It suppose to have no colonies if DpnI works completely because the control after PCR has only methylated plasmid (no new backbone with mutation and no methylation) with the same amount backbone in experimental samples. If it gave a few colonies (sometimes DpnI is old and does not work perfectly), you can consider it as background and minus the colony number in the control for the experimental plates to how good quality of PCR mutagenesis was. Don't worry for wrong pick-up colonies because you can check it later by sequencing.
Hope it helps. Good luck with mutagenesis! :)
Hello Monika,
You set the control at the same time when you run PCR for site directed mutagenesis. The control is the plasmid without the primer (the primers have mutations). You use the same amount of plasmid with the experimental samples to run the same protocol and treat DpnI. Then perform transfomation. It suppose to have no colonies if DpnI works completely because the control after PCR has only methylated plasmid (no new backbone with mutation and no methylation) with the same amount backbone in experimental samples. If it gave a few colonies (sometimes DpnI is old and does not work perfectly), you can consider it as background and minus the colony number in the control for the experimental plates to how good quality of PCR mutagenesis was. Don't worry for wrong pick-up colonies because you can check it later by sequencing.
Hope it helps. Good luck with mutagenesis! :)
I will take out 5 uL of my SDM reaction immediately before adding DpnI, and then I'll transform it at the same time as 5 uL of the DpnI reaction product. This is really only a control to give you the confidence to go forward.
I typically perform the SDM reaction in 20 to 60 uL (total volume will depend on whether I'm confident in the reaction and whether I need to use a gradient). Once I have gel verified that I have amplified the plasmid, I then digest with 1 uL of DpnI. Then I will transform my E. coli with about 5 uL of my digest.
If I need a control, I will remove 5 uL after the gel verification but before the DpnI digest. When it comes time for the transformation, I'll then have two 5 uL samples to put into my bacteria. This will tell me if I've actually amplified the plasmid because I should see a difference between the two transformations. It's good to find this out before you go through the trouble of miniprepping and sequencing.
Hi Eric,
Thank you for your explanation. It's really helpful. May I know how do you do the gel verification for the amplified plasmid? I'm afraid I wouldn't be able to differentiate between the parental plasmid (used as template) and the amplified plasmids. Thank you
Hello Nurshariza--
You shouldn't have much template (no more than 50 ng, though some protocols call for less). This may show up on the gel, but there won't be much of it; if your amplification worked, you'll have a strong band. If you're really nervous, remove an aliquot before you do your SDM reaction and load it on the gel.
Also remember that your product will be linear on the gel, so its band will run at the right size. Your template will give one or two bands for supercoiled and nicked that likely won't correspond to the correct size. It's fairly obvious when the reaction works.
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