I am trying to delete 30nt regions of a plasmid but have been unsuccessful in my PCR using overlap extension PCR. My primers range in size between 23 and 36 bp in length. I fear that my deletion primers are too small and cannot adequately flank the region that needs to be deleted. I was thinking of making the primers 60nt in length so that they could efficiently cover the region to be deleted. Is this a good size for these primers?
I have done many side directed mutant construction. As far as I remember I used b/t 45-50nt long primer. What method do you exactly use for construction? Do you have a publication about it? If you can share with me, I like to make more suggestion. It is a tidies process, and good luck...
When I modify genes, I use as a general rule primers annelaing with the target sequence 15 nucleotides before and 15 nucleotides after the region to be modified, that means at least 30 nt length. Making longer primers do not necesarily mimprove results.
I always try that the primer ends are G/C rich. Sometimes I make longer primers just to fulfill this requirement. Hairpin formation should be avoided by primer design.
There are several options for such a deletion ()Kunkel mutagenesis,quick change mutagenesis . You can try any of them.
Hi Charles,
i recommend more than 45 nucleotide with GC more than 50% in yr primer.
Always end your primer with G or C in twice or more.
I experience more success rate when i use single primer model than two primers for creating mutations. I have done following single primer to delete more than 100bp.
Hi Karthikeyan,
I need to delete a 137bp fragment from a plasmid; I'm interested in your approach, could you kindly send me your protocol?
Hi Charles,
Did you get it? I would like to delete a fragment from a plasmid too but I have no idea how many nucleotides I could delete using a Mutagenesis kit.
Thanks! :)
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