Problems ligating shRNA primers - Sequencing reveals damaged DNA (May/18/2010 )

Dear Scientists,

I am trying to make 29-mer shRNA loops from complementary oligos, but having no success.

I have created the duplex by cooling from a high temperature and confirmed all is well by running on an agarose gel and purifying the ds bits (being careful with UV exposure). I've then used T4 kinase to phosphorylate and used Roche Rapid Ligase to try to complete the circle into Promegas siStrike vector. The final concentration of DNA in the ligation mix is 5ng/ul, with the insert being 200pg/ul of this.

There were mostly empty colonies, but colony PCR helps to choose the right ones. The problem I'm having though is that the positive colonies I find appear to have damaged DNA in them. I've never seen anything like it really, the insert is only half there. Admittedly this is just from two positive clones, but one of them was missing the first 20 bases of the 70 base insert, and even more bizarrely the other was missing the first 20 too, but was inserted in the reverse orientation. These were from two completely different shRNAs!

I will repeat all this of course, but if anyone has any ideas why I'm managing to lose the first 20 bases of my inserts I would love to here them.

Thanks,
IC

Hi IC,

Yes, what you got seems bizarre and not very common.

One possible cause is quality of the oligos synthesis is poor, resulting truncated oligos. Try a different vendor or resynthesze them. Desalted is good enough.

There is no need to phosphorylate the oligos before ligation.