This primer will be used for colony PCR. My lecturer suggested that I choose a region that includes both the backbone and insert gene so that it can ensure specificity. The plasmid is pET-22b(+), and the inserts are around 1353 bp long, but the amplicon will only be 700 bp. And what is the best tool to design this kind of primer? Thank you in advance, your suggestions and insights are highly appreciated.
Dear Virania Putri
what do you mean for primer lenght 700bp? Do you mean the lenght of the amplified fragment?
in the following video you can find some informations about the PCR colony screening
https://www.blogger.com/video.g?token=AD6v5dzrZXbjCrsrnUvj5lmNOcOD_G10hIZc21CiOmmZoA6maPuNq-rPahUPP6mkAmCN3gEo8TW8PYjV4y-HkExg9cZ0gYqMyUGgoMr45rgDOWushBW8LocpxX5tGVjD633sQB3lRuVe
unfortunatelly there are few informations about the primer desing (just a simple picture around minute 1'00 but i hope that it can at least clarify a little your doubts.
in pet vector generally you can use T7 promoter or T 7 terminator primers as annealing primers for the backborn, so theoretically if the annealing temperature are not so different you can use:
choiche 1:
T7 promoter coupled with the reverse primer that you have bee use for the cloning of your gene
choiche 2:
forward primer that you use for the cloning coupled with the t7 reverse
in this way you will amplify a fragment little bit more longer that your insert.
best regards
good luck
Manuele
You should check your notes from class.
Primers are typically about 20 nucleotides in length, not 700.
Your professor is wanting you to design primers that will amplify just part of the insert and not the entire insert. One primer will be in the plasmid backbone, the other will be in the insert.
That should be more than enough for you to figure this out.
Katie A S Burnette I am so sorry, what I meant by 700 bp is the amplified fragment, thank you for your advice!
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