PEG 4000 for ligation - (Sep/04/2008 )

I read in this forum that PEG 4000 might help increase ligation rates? I looked it up in the SIGMA catalog and there seems to be different kinds and percentages of it? Maybe someone could tell me which one they use?

Also a TBE buffer inhibits ligation and its better to let the gel run in TAE? Why?

I think I might have to try a triple ligation with fragments that are 1kb, 1.5kb and 4.5 kb, I cant seem to douple digest my vector properly with Xho1 and Bgl2. Could someone tell me how much of each I should put in a ligation reaction? The final volume should be 20 microlitres right?


thanx!

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A triple ligation would be much harder, especially considering that you seem to be new to molecular biology, I would suggest that you exhaust every possibility of doing it another way first.

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I have to agree with Smu. A three way ligation is more difficult than a 2 way ligation (although not by much, if you are secure in your skills.). The molar ratio used are 1:1:1 for vector:insertA:insertB...etc.

PEG does help with difficult ligation (especially useful with blunt end ligations). However PEG (also found in quick ligase) does not work well multi-ligation reactions. Better to stick to normal T4 ligase buffer when conducting multiligation.

I would follow Smu suggestion and exhaust all possibilities. Have you considered increasing the amount of time for digestion? Increase the digestion mix's volume?

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I did increase digestion, I started out with 5 micrograms and did two parallel digests, one with Xho1 and one with Bgl2, 20 units each, after two hours I took a sample and loaded a gel while the samples incubated further. Gel pic showed uncut vector for Xho1 and none for Bgl2 so I then added a lil more Xho1 for 30 min to the Xho1 digest and added 20 units Xho1 to the Bgl2 digest. After the 30 min I then added Bgl2 to the other digest.

I had digested in 100 microliters and I only took 40 of each double digest and loaded a 0.8% gel, I only used 10 microliters per gel pocket. I ran uncut vector, my old double digest, and samples of the digest with Xho1 and Bgl2 alongside my double digests.

Uncut ran further up than any other

single digests underneat that

my old double digest (1h 37 C in Buffer D using Promega enzymes) ran below that

and my new double digests also ran below but the bands were so thick that it didnt look right. The band was so thick it went from were my old double digest was all the way up to single digests.
Also the stuffer sequence that came out was way fainter that the supposedly double digested vector,
it looked like not everything had been digested properly. For the amount of vector that I saw in the thick band I should have had more stuffer sequence coming out.

So it looks like a problem with the double digest and the amount of material that im digesting.

Its weird.

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Have diagnosed the problem, good job! There is too much DNA and the enzyme are not cutting fast enough for the time given.

You might also want to do a small digest, to see if the XhoI site exist and can cut first. (Seems you have already done this)

I think this problem can be solved by either cutting down the amount of DNA digested, or digesting the vector overnight (in a double digest). Keep the 100ul volume. Do not increase the amount of enzyme, keep to 1-1.5ul. Too much enzyme and the glycerol the enzymes comes in will inhibit the reaction. Inhibition comes when volume of enzyme used is about 5% of the total volume of the digestion mix.

Take a piece of tape and tape it over several well teeth(6-7), to make one single giant well. That should solve the well overloading (the spread you have observed).

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hehe I made a huge gel pocket and loaded 100 µl of a 1:10 dilution of what I digested yesterday. Then let it run for like 3 hours.

But ill also do another double digest with less DNA and my new enzymes hehe hopefully I wont get this again:

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