Site-directed Mutagenesis

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ProteinQuest
ProteinQuest's picture
Site-directed Mutagenesis

Hello everyone, I need help on finding out why I'm getting wild type DNA sequence even though I used a mutated template to introduce one more mutation. Since I had no luck on using quick mutagenesis kit, Here is what I did. I made 5' end insert and 3' end insert separately using mutated template with appropriate primers, and then, used them to make a full length insert. I ligated the insert with vector that was digested with the same restriction enzymes. All steps were checked by running agarose gel to see the size of the inserts, template and the vector, and products from each step were purified by Gel extraction. E.coli transformation (XL-1 Blue) was carried and products were mini-prepped before sending for DNA sequencing.
Please help me as soon as possible. Thanks

Jason King
Jason King's picture
Is this right?

Is this right?

You introduced a mutation in to the centre of your insert by performing two PCR reactions in which the reverse primer for the left side overlapped the forward primer for the right side? And then you used the outermost primers again, together with both individual PCR products to produce a full length PCR product?