I am trying to introduce SDM for a gene on a plasmid. After Pcr (with HF Q5 polymerase and primers designed by NEB website the length of one of them 19) , Dpn 1, ligation, transformation and miniprep were done. Then I sent the clean miniprep to sequencing after adding the appropriate primers. The sequence result completely aligned with the gene except in one position where my mutation introduced but in position 324 instead of introduced at position 331
Do you think I should design the primer for SDM by myself to be longer especially I am using 3 plasmids 7.5Kb, 9.5kb and 11kb and the length of the gene 3 kb.
I think you should consider designing the primers yourself as the correct mutation was introduced, however, at an incorrect position. Aim for SDM primers of approximately 30 bp in length with your mutated site as close to the center as possible. While it is acceptable to make primers a little longer or shorter as required, there should be a minimum of 12 bp either side of your mutated site.
Mariam Abdelmalak , The whole plasmid PCR mutagenesis requires the properly designed primers which involves several critical factors such as lengths and nucleotide composition of sequences flanking the mutagenic site, the GC content and melting temperature (Tm) of the primers, etc. Generally, the primer length should range 25–45 bps. So, I agree with Tushar Chakraborty that you should re-design the primers for the successful SDM. You need to be very careful about any mismatch or flanking sequences.
thank you very much Tushar Chakraborty Shaista Bano Michael Halim
Tushar Chakraborty Shaista Bano I have designed new reverse primer with the mutated nucleoide in the middle, the length is 31 instead of 19, CG content 51.6%, 3'ends with GC. I want to evaluate the presence of the 2 primers together (forward and reverse) with my sequence. I have used SnapGene before but unfortunately my free trial is expired. What do you recommend instead?
Mariam Abdelmalak As you mentioned that your SnapGene free trial has expired, you can try NCBI Primer Blast.
Dear Mariam Abdelmalak , Check the following links, if these are helpful,
http://www.bioinformatics.org/primerx
https://www.agilent.com/store/primerDesignProgram.jsp
http://www.oligoarchitect.com/LoginServlet
http://nebasechanger.neb.com/
Thank you very much Shaista Bano Tushar Chakraborty
In case of complementary primers, does SDM work?
I mean although they are complementary, instead of binding to each other, they bind to my template.
I can highly recommend this protocol here for site-directed mutagenesis:
Liu H, Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008 Dec 4;8:91. PMID: 19055817
- They use only partially overlapping primers (and have an additional 3' overhangs) that are supposed to promote/favour primer-template binding over primer-primer binding.
- Also the primer/temperature design makes sure that primer anneal to the original template plasmid (circular) and not to the PCR-product from previous rounds (nicked).
Just try it out; All you need are a pair of longer primers (~45); Proof-reading polymerase, DpnI and E.coli you already have ;-)
Since there are 1000 ways to calculate the annealing temperatures: I used the the "#2, salt-adjusted" one from "Oligo-Calc" (http://biotools.nubic.northwestern.edu/OligoCalc.html). It assumes concentration of monovalent ions of 50 mM (which was quite close to my PCR buffer, dNTPs, polymerase).
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