problem with the MCS - I can't use it. (Apr/04/2006 )
I'm drawing a blank with my experiment here, and would love to have some advice:
I have a plasmid, which need to insert a gene into.
The problem is, the MCS has only 4 RE sites, and I can't use any of them. Either they cut the plasmid twice or more, or they'll cut my gene.
I, apparantly, need this plasmid, because it has the promoter, SV40, and Poly A.
So, I was wondering, what would you do?
I was thinking, I have another plasmid that has the Sv40 and Poly A, and i should cut out the promoter from plasmid 1, and stick it in plasmid 2.
Which brings another problem: The enzymes used to cut out the promoter don't sit will in plasmid 2's MCS. It'll mean i have a problem putting my gene in the right spot.
So, I was thinking, designing some primers to amplify the promoter out, with the restriciton sites that make me happy.... but then, there is no sequence for plasmid 1, and i don't exactly know how much of the promoter is in plasmid 1, so i could really stuff of the reverse primer...
why dont you design you own MCS, with the suitable sites, and paste the polylinker in the MCS in your vector?
design a mcs with required restriction sites keeping an infram translation (if required). Designe two oligos ,that contain these sequences, and anneal them should do the job (oligos would be designed to anneal in a way their extremities don't need to be digested).
Hi, I am not sure if perhaps this might work. If I am reading your post correctly, there is a RE in MCS that cuts your plasmid once, but it would also cut your gene. So what I would suggest is to use the first plasmid, and cut it with the enzyme that cuts it only once (I am assuming that this enzyme maybe cuts your gene), then N-fill it, make it blunt. Cut out your gene, and make it blunt. Ligate the two together (blunt end ligation), and then check your clones for the orientation that you need it to be in. Technically speaking there should be 50% chance that your insert will get into the desired orientation. How do you check for this? Cut with the appropriate RE. Good Luck,
have you thought of fusing your promoter to your gene by PCR. You can amplify the promoter, then the gene and then mix them together and do an other PCR for amplification of the entire fused product. There is more to it than what I have just said, your primers have to have overlapping regions for it to work, buts its all in the primer design and not hard. There are papers out there on the method..I can find it should you be interested. You could then clone the fusion product into pGEM or pTOPO and cut it out with REs that would allow for it to go into your special vector.