how can I shift the frame? - please help!!!! (Nov/01/2006 )
i constructed the vector and it came out that i made mistake in one base and now GFP is not transcriped (it is after my insert) because of this. 
i only basically need to remove one base from the vector...it is possible? i checked all the RE sites that can effect the frame shift again but there arnt any... all the mutagenisis techniques seem super difficult. please help me! i have no time to construct the new one.
this is a difficult one.
Could it be possible to post the sequence, map and anotated diagram of your sequence? Genebank or embl format would be fine. Point out the place of the frame shift.
I don't know if a repair by fancy restriction digest footwork is doable but with that bit of info I'll give a wack at the problem.
Although it might be easier to simply invoke "Unlimited Ligation Works" and rebuild the plasmid in one multiway ligation step. Especially if nearly all the fragments are available. How many parts were required to build this plasmid? Alternatively do a mutagenesis, which would be easy if the plasmid isn't too big.
thank you for your reply. however i think it is difficult for me to post the sequence since so many cuts and insertions, just dont know how to 
. i have checked all possible RE sites to shift the frame back it seems like none so i think im left with just cutting the GFP out of the vector (since now it is not GFP anymore and i dont want some weird protein hanging next to mine). but the question is there are sites and i can perfectly cut, blunt end and ligate back but im worried about the stop codon. i will cut out this one too. there is a terminator at the end. will it be ok? sorry for changing the topic a little.
i am not sure what you are describing. Do you intend to cut out your insert...blunt end it and religate and wondering if the ends would be okay? Well if you are blunt ending by filling in the ends with klenow, the ends should be okay. I am a bit jittery using nucleases though, overuse can lead to a "BAD END" senario.
I am not sure what is happening to your plasmid. I am not sure what you mean by the stop codon. Could you draw a picture? Do you have two protein ORFs on the same RNA transcript, co expression of GFP and your protein. If your protein has its own ribosomal binding site, I would guess that it would be okay. The ribosome ignores all codon until it finds the first ATG codon. (If I remember my stuff correctly) before starting up the translation.
Why is mutagenesis difficult? Seems to me that desiging a pair of primers with an extra base (or a deleted base) should be possible?
Agree with Vairus,
this is what i would also do, design primers with a deleted base (two complementary primers of 30 bases, 15 bases on each side of the deletion). check that the mutation doesn't give you a restriction site, especially with the RE you would maybe use.
I did successfully "quick change mutagenesis", using Pfu turbo polymerase,
use 50ng of DNA template,
95° 2min
95° 45s
50° 1 min
68° 10 min (depend on the size of the vector)
14°c hold
repeat steps 2-4 20 times
you can check that the PCR worked by loading 5µL out of the 50 µL of the PCR reaction, on a gel, and also the parental plasmid (5 ng) to be sure that the DNA you see is the amplified DNA. If you titrated well your DNA, you should not be able to see non amplifed DNA (5 ng) on the gel.l
then digest the parental DNA with DpnI (I used 1 µL that is 10 u) 1hour 37°C, and transform bacteria with 1 µL.
good luck
Missele thanx a lot for the advice. the thing is that i never used mutagenesis so im a bot worried about it.
good luck
what do you mean by that? how can DpnI digest if i dont have its site in the DNA?
and why to do that? another stupid question sorry, will fusion do for mutagenesis? (or is it phusion?) or only pfu should be used?
it seems i can shift the frame by digesting with 1 RE and then blunt end and ligate it 
im genius!!!! i hope it works. will let you know in case it doesnt 
thanx guys!
Nothing much.
Both Phusion and pfu are high fidelity enzymes.
Phusion is pfu fused with a double strand DNA binding protein, thus the polymerase has better processivity (then pfu) as it doesn't fall off easily once it starts.
Just work out the repair on paper and computer, how does the fill in affect the codon sequence - protein sequence. How much of the protein is frame shifted before it gets back into frame