How do I check frame? - (Apr/24/2006 )

Hello All,

I am being asked to make fluorescent protein construct of promoter region of this gene. I am not molecular biologist so please bear with my stupid question.
I am being given a plasmid in which this fluorescent protein is already there. All I need to do is, PCR the promoter region 3kb or so and insert in this plasmid just before the fluorescent protein.

I was wondering how do I check that the PCR product of promoter region is in frame with fluorescent protein???

Thank you

Anybody that knows, please reply!!

Everybody tells me to check the frame while designing the primer but they don't tell how to check!!

Well, 3 bases build one amino acid. The 3`end (f.ex. stop codon) of your new gene and the start codon of the fluorescent one need to be in one frame:

`TAG``ATG` --> `stop``start`

So, I guess you have a restriction site in front of the second gene and a few extra bases (3 or 6):

TAGXXXRRRRRRATG

Stop and start are still in frame...

Then you need to design your primers:

For the forward primer you take:

3 extra bases, 6 bases for the restriction site and the first 18 bases or so of your promoter region

For your reverse primer you take (I mean from the 5` end of the 2nd DNA strand):

3 extra bases, 6 bases for the restriction site and the last 18 bases of your promoter (5` --> 3` of the 2nd strand)

YOu come up with this:

.....PPPTAGRRRRRRXXXXXXRRRRRRATG

To sum it all up: between the 3`end of the promoter and the start codon of the fluorescence gene you need to have a number of bases that can be devided by 3.

oh and remember to check that the restriction sites you design in your primers are unique and do not occur elsewhere in the plasmid