Double Blunt Ended Ligation? - (Sep/26/2006 )

Hello again

More problems: I digested my vector (~3200 bp) with the restriction enzymes SspI and EcoRV. The reaction was done as a double digestion in appropiate buffer and temperature. This yielded a linear backbone of ~2800 bp and a ~400 bp dropout. The linear backbone was gel purified and dephosphorylated. My exogenous insert was also digest with SspI and EcoRV ~2900 bp.

Using Roche's Rapid Ligation Kit, I have failed to ligate the two. I did try a 1:3 ratio of vector to insert and transformed <10% of the total volume of competent E. coli cells.

Anyone any ideas?

[Hi!
Why don't you try with ratio 1:10. I haven't used this kit, but I tried to ligate with Takara kit, it was written to use 1:3 ratio, for 30 min at 16. It simply didn't work. So I did it with 1:10 ratio vector:insert and ligation overnight.
There is a formula for how much insert you need (in ng)= (10*vector(ng)*insert size (bp))/vector size (bp).
Hope it will help you!

Here are the standard answers,

Try an overnight ligation. Although it is a quick ligation kit, a double blunt end ligation is far less efficient.

Try ligating at lower temperatures... try 16 Celsius if the experiment was done originally at room temperature. try 4 Celsius if the experiment was conducted at 16.

Are the ligase buffer and ligase in good condition? Make sure none of these two have gone off.

As already stated try using more inserts, however be aware that the probability of double inserts increases, so do the appropriate RE digest to differentiate single inserts and double inserts.

Thanks for all the suggestions

Overnight ligation at RT and 16oC did not yield any colonies.

Have tried vector : insert 1:1, 1:3, 1:5, 1:10, still no joy (Have gotten some colonies back but these did not confrom when digested with specific restriction enzymes).

The Kit Im using is my own (nobody else uses it) and opened in the last 3 weeks.

When run on a 1% agarose gel with EtBr, the vector and insert appear to be correct molecular weight etc.

Was thinking of the reversing the ratios, giving a larger proportion of vector to insert in the hope of getting one successful colony.

Anyone any opinions??

How are you purifying? For example, are you exposing the gel to UV for cutting the bands out? Exposure of your DNA to UV can damage the DNA. If you are, try not doing this by running a bit of your sample next to the lane where you intend to cut the band out. Cut the gel and only expose the lane with a bit of your sample. Mark on the gel where your hand on interest is and line it up with the other half of the gel..then cut your band from the other lane and purify.

Blunt end ligations are tough and your doing a hard one by ligating to large fragments together. Therefore, ligate for 16 hours to maximise your chances of it working and use very high efficient competent cells. You can either purchase highly efficient competent cells or try an electroporation.

only have 2.5 ideas left

1-Once all the ingrediant are added, decrease the volume via EtOH percipiatation. (And resuspend to maybe 9ul, heat to 65 for 5min, cool and then added the appropriate buffers & ligase) And ligate at 4 celsius overnight.

1.5-Don't dephosphorylate the vector (and try to ride out the large number of false colonies) Might you have colour testing? Use something like 10x insert for this reaction.

Transform as much of the ligation mix into the cells as can be safely done.


2- Redesign the insert. Ie buy primers and amplify the insert RE sites that make with sticky ends which are ligateable to the final vector. (This may well be the fastest method.)

Best of luck.

I have done double digestion, cohesive ends, but might help.
Vector is ~3kb and insert ~2.3. Did different ration and 1:3 (insert to vector) worked beautifully. It was 2hr ligation at room temperature.

Hi

I think perneseblue's suggestion, that don't dephosphorylate vector and add 10X or higher insert, is alright. You should give it a try. You could try using different ligase as well, just in case that's the source all along.

Worst come to worst, as suggested, you should design primers with RE sites that yield sticky ends.

Cheers