QUestion on subcloning a 80bp product from TOPO vector - (Mar/28/2008 )

Hello everyone,

I have a 80 bp product cloned into a TOPO vector. I need to sub-clone it into another vector....

When I do a restriction digestion of 2 ug of TOPO vector with insert, I am not able to visualize my 80 bp product (because of its size) in the gel. So, I am unable to do a gel elusion and subsequent ligation.

I desperately need to sub-clone it with in the next 1 week.....any suggestions?

Thanks in advance.

-micron-

QUOTE (micron @ Mar 28 2008, 11:37 AM)

you could try to do PCR using M13F/R primers, cut with your enzymes and then perform the ligation. It'd be kind of small though, so watch your pcr conditions and don't confuse it with primer dimers.

-smu2-

You should be able to visualize an 80bp insert if you run it out on a high percentage gel (maybe 4-5%). There are ladders specifically for working with small fragments. Personally I like the NEB 100bp ladder or better yet, Fermentas has a line of ladders (O'RangeRuler) which is either from 10-100bp in 5bp increments, 10-150bp in 10bp increment, 20-300bp in 20bp increments. You just need to make sure you stain and destain well to clearly visualize the fragment.

-rkay447-

Thanks smu2.

Thanks again rkay447. Do you think that if I digest may be 5-6 ug of the TOPO vector with the insert to be able to visualize the 80bp band, gel purify it for subsequent ligation, that will be sufficient?

Thanks,
Micron

-micron-

Hey,

I agree with rkay447. You should be able to see it on the gel.

You would see 50-100 ng of your 80bp band on the gel, for sure. You can calculate how much DNA you have to cut in order to have that about of the band of your interest (sorry, I don't know the size of the vector).

When you prepare the gel, use a comb with narrow teeth. It will be easier to see the band on the gel even though you don't have much DNA.

Cheers

-zek-

i think 80 bp is kinda small. maybe send for sequencing?

-timjim-