Multimerization Troubleshooting: No Luck in 2-3 Weeks

[karthik3k]

Multimers :((

I have been trying this thing for past 2-3 weeks with no luck :((



I am trying to make a multimer from an oligo.
This is how i did:
- Annealed the oligos
- Phosphorylated the sample (5' ends)
- ligated o/n

when i ran it on agarose it shows only a dimer. what can be the reason ?
any ideas ?

Should i use some different protocol ??

 

[Monique]

When I did ligations, I could see a ladder. My fragment was in a vector, which I excised with restriction enzymes. The result is sticky ends which ligate much better than blunt ends.

So either your ligation reaction is not efficient, or you are making circular DNA molecules. The get more linear product, you should ensure that the concentration of DNA in your ligation reaction is quite high. That way the DNA is more likely to come across a new DNA molecule to ligate to, than it is to its own free end. Another problem I see is that you use very small oligos. Mine were 100 bp, so the ends are further apart, thus less circular products.

 

[karthik3k]

k.

do u have any established protocol for this kinda problem ?