how to ligate a gene repeatedly - (Aug/08/2007 )
I want to express a poly peptide, is there any protocol can ligate the gene head to tail repeatedly?
I suppose you could just set up the PCR such that you can generate multimers upon ligation. The easiest way is to use a single restriction site at the termini of the primers. Then, after PCR and digestion, ligate the products together under a range of conditions (so you generate a number of different length products), gel purify the product with the desired number of repeats, and clone this into your vector.
Naturally, if you do try this, you'll have to make sure that each single unit has a stop signal, or better a number of stop signals.
Problems: When the mRNA gets made, there is a natural overrun (but I don't know if you can guarantee how far this will go). Depending on the size of the poly peptide, you might get two constructs, you might get 5.
Suck it and see if it's an orange or a lemon.
Concatermerization by ligation should give you repeated sequences. You can purify the product by gel electrophoresis for fragments with the desired size. Check ref for such works. I know people have made artificial silk proteins this way.
Basically, you design an oligo or isolate a restriction fragment that has complimentory ends ( for example, both ends have identical half site for one enzyme, ECoRI, or one has Bam H1, the other has Bgl II) and ligate itself over and over using T4 ligase.
is there any paper or protocol concerning it? please give me the link.
I've never heard of such a protocol. Just treat it like any other ligation. When you get things working, you could post the protocol here, and we'd all be in your debt.
Make your primers with the same RE site, as genehunter said.
two primers have the same RE site, is that mean the ligation has no direction?
Yes, which means that, statistically, you will have half of the constructs the right way around, and half the wrong way around. You can check which are right by doing a bunch of small-scale expressions, and selecting the ones that work, then sequencing those plasmids to confirm, or you can do a pile of sequencing reactions, and select the ones that are the right way around, and express using them...