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I am trying to use primer pairs with overlapping and non-overlapping regions to create deletions within a protein sequence (https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91).

I aim to delete at least 10 amino acids within a region of choice. I did PCR using the primer pairs I designed and out of 4 transformants I screened, I got one deletion I was aiming for.

I then did the same for deleting another region and I didn't get any required deletions. Vector size is ~5kb and the gene is 1.6 kb. I do Dpn1 digestion for 2 hours using 1ul of enzyume. My questions are these: a)Is improper Dpn1 treatment the reason for failing to get enough transformants? b) Since I am doing full plasmid amplification here, are there any things I should keep in mind while I perform the reactions?

I am terribly sorry I am not being specific anywhere. I am quite new to everything described here and has only started to get a hang of things. Any help would be greatly appreciated. Thank you!

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Malka, you are on the right track with Site-Directed mutagenesis! I agree with previous answer about NEB method and their kit. If you can, please-please, get yourself their product here. https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit

I have spent a lot of time with kits from different vendors, and, in parallel, attempted to combine enzymes and substitute reagents in troubleshooting steps. All my efforts ended up as half-measures and a ultimately a waste of time. In theory, yes, you can make it all work with off the shelf reagents, but (unless you get lucky) your substantial effort and investment of precious resources (i.e. competent cells, and time) may cost a lot more in the end.

  1. Go for the Q5 Neb kit (all included)
  2. Design primers using their software tool (i prefer the older version) https://nebasechangerv1.neb.com
  3. Use exactly as directed. And drink coffee, read papers being confident that in two weeks you will get exactly what you need.

Feel free to ask any more questions about this.

To answer specific questions you ask above: (a) No, extended DpnI treatement should not reduce the number of transformants. (b) Yes! Make sure that your extension step is long enough to amplify the entire plasmid accounting for its size. You can run a quick gel to check the size and amount of your PCR product before proceeding to the cloning step.

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10 aa or 30 bases is about the limit of traditional quick-change style systems where you have overlapping or completely complementary primers. Kunkel's 1985 paper1 used complementary primers, but variants have been developed since. The limit is partly because generating primers that don't incorporate large chunks of the target tend to form weird structures in the PCR and not anneal well; it also costs more to have primers that are about 50 or more bases long for such a PCR, compared to conventional 18-25 bases.

There is a variant of quick-change where you basically generate primers that are "back-to-back" so that you amplify around the plasmid, which doesn't limit your size of deletion. In this you use the primers to flank the region to be deleted so that you generate a linear transcript that incorporates the plasmid. New England Biolabs (NEB) has a good description of the various types here.

For these types of protocols you do need to phosphorylate the product (using a kinase such as T4 polynucleotide kinase), ligate the product using a DNA ligase and then DpnI treat the product before transformation. These can be bought as a kit or as individual components, you may even have some of these in your lab right now if it is a lab that has done cloning before.

Ref:

  1. Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92. doi: 10.1073/pnas.82.2.488. PMID: 3881765; PMCID: PMC397064.
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