how to make a in frame insertion ? - (Apr/17/2008 )
I am new to molecular biology and especially cloning. I want to make an in frame insertion of a gene into a vector. I have a couple of questions on this:
Does making in frame insertions depend on design of primers ?
As far as my intuition goes on the molecular biology of PCR, the overhangs that are part of the primer design + restriction site regions have to add up to be a multiple of 3 to be in frame with the coding region. Is this correct ? I have drawn a diagram of a typical primer below:
OVERHANG RESTRICTION_SITE ATG rest of the coding region .............
If anybody could give me a little bit more information on in frame design of primers, I would really appreciate it. Even diagrams of working primers would be very helpful.
Thanks in advance,
vjszq4.
Does making in frame insertions depend on design of primers ?
As far as my intuition goes on the molecular biology of PCR, the overhangs that are part of the primer design + restriction site regions have to add up to be a multiple of 3 to be in frame with the coding region. Is this correct ? I have drawn a diagram of a typical primer below:
OVERHANG RESTRICTION_SITE ATG rest of the coding region .............
If anybody could give me a little bit more information on in frame design of primers, I would really appreciate it. Even diagrams of working primers would be very helpful.
Thanks in advance,
vjszq4.
You've got the right idea. The overhang does not need to be over 5bp for most restriction enzymes. If you clone into Topo-TA vectors (invitrogen or clontech) you don't even need to cut and ligate. just mix pcr frag and vector, then transform.
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