Oligo-cloning - (Aug/06/2005 )

Hi all,

I am cloning the 5'UTR of my gene of interest in pGEMT easy and I have questions about how to go about it.

Here is some information regarding it. The UTR is synthetically synthesized and is of 87 base pair. I have both the sense and antisense strand.

I require to anneal them both and add A tail by Taq Pol.
Could you suggest the procedure?

And does the annealed product need to be purified before cloning?

Thanks

Mili

if you have both sense and antisense strand, you can try annealling directly. the procedure is as follows:
anealling system: sense strand, antisense strand, add some sterile water.
95 cen for 6 mins, then cool slowly until rt.
you can use a big container to heat water.
if you only use it for clone, you may directly use it, not need recovery.

of course, if the annealling doesn't work very well, you can add some buffer, the 10*annealling buffer is 10 mM MgCl2, 200 mM Tris-HCl, pH 8.0, but you should recover the fragment from the gel before you ligation.

cheers,
braveheart

Hi Mili,

After they're annealed you just need to A-tail the product before cloning into a T-vector. This method is straight from the pGEM-T/pGEM-Teasy booklet:

1) Start with 1-7ul of DNA
2) Add 1ul Taq DNA Polymerase 10x reaction buffer with MgCl2
3) Add dATP to a final concentration of 0.2mM
4) Add 5U Taq DNA Polymerase
5) Add deionised water to a final reaction volume of 10ul
6) Incubate at 70C for 15-30min
7) Use 1-2ul in a ligation reaction with your T-vector

I've use this successfully for cloning PCR products made with proof-reading enzymes (like Pfu) and had no problem. (5U of Taq is a bit of an overkill, so you can use less if you like).

Because your product is small, you should try a variety of vector:insert ratios for efficient ligation.

Good luck!

Nicole