Clonning problems - my ligation didn't work (Oct/24/2006 )

Hi everybody,

Maybe somebody can help me with this.

I'm trying to clone a fragment of about 1.7 Kb digested with Bam HI and Hpa I (blunt) in a vector of 5.6 Kb digested with Bam HI and Eco RV (blunt). I already did 2 ligations and they didn't work. At first I thought it was my plasmid was not well digested. Since both sites were too close in the polylinker I could not see parcial digestion but after doing digestions separately and purifying after each one now I think that's not the problem.

- Could it be in my ratio vector:insert, I normally use 1:3 but I don't know if since insert is so big that still works?

- It is maybe ligation conditions? IO did ligation at 4ºC avernight. I have a blunt end cutted with 2 diferent enzymes but Shouldn't it be a problem?

Well I hope somebody have some advises or sugestions.


Thanks a lot

I usually leave my ligations at RT for 1 -2 hrs before transforming them.

hmm....

1:3 ratio is okay. The insert and vector aren't actually that big. If you have enough insert you could increase the vector:insert ratio to 1:5. Burt is should have that big effect.

The ligation conditions should be okay. Though for an overnight ligation reaction I would run it at 16 Celsius.


What is the formulation of your ligation mix? How much DNA are you ligating?

Do you gel purify your DNA fragments? Is you be aware that any residual wash buffer and gel melting solution (the orange coloured solution) will severely interfere with subsequent ligation reaction. I leave the wash buffer sitting on the filter column for 2mins before spining it down.

Is are any of your DNA fragments in TE? EDTA will also kill the ligation reaction.

Don't over exposed your DNA bands to UV light when you cut it out of the gel slab.

Be aware that T4 ligase and T4 ligase buffer do go off when exposed to freeze thaw cycles. T4 ligase goes off the fastest. The T4 ligase buffer has a strong smell of DTT (Dithiothreitol). Get a new vial of T4 ligase buffer, open it and memories that smell. If the smell that smell is gone so is the buffer. Always aliquote the ligase buffer.

After the ligation is complete, you could run a gel on the ligation mix. Run maybe 30% of the ligation mix, you should see bands of higher molecular weight if the ligation reaction is successful.

well I will try increasing vector:insert ratio. As for the ligation mix, I use about 200ng of DNA in total (vector + insert), I could use more i have a lot, just how much is the maximum or minimum?

I purified my DNA's from gel with the qiagen gel extraction kit and I solved them in water althought I have not tried that of letting the washing buffer for some time in the column.

Thanks for your other sugestions I will take them in account for my next try.

try to start with 20ng of vector and calculate ur insert from this. U would end with 30-40 ng of DNA at most. This would work for most cloning steps.

There is no clear levels of DNA which is max. or min.. Just from experience.

200ng - total DNA - is too high, but I have used it for some resistant cloning steps.