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No band in the gel after ligation - (Mar/17/2009 )

hi
I prepared a ligation mix of 15microlitr. My insert was of 1640 bp and vector of 4000 bp. after incubating in RT for 1hr, i checked the mix in 1% agarose, butni didnt find any band except the marker...Can anyone give me a goodsuggetion.


priya

-priyak-

Hey

Did the elutions work? Did you check the fragments on a gel before setting up the ligations?

Best
TC

priyak on Mar 18 2009, 07:25 AM said:

hi
I prepared a ligation mix of 15microlitr. My insert was of 1640 bp and vector of 4000 bp. after incubating in RT for 1hr, i checked the mix in 1% agarose, butni didnt find any band except the marker...Can anyone give me a goodsuggetion.


priya

-T C-

TC is right.
Did you gel purify your insert? Did you clean up your digested vector using either gel purification or PCR clean up kit?

Once you are sure that you are using right vector and insert, then setup ligation in only 10l using 1:5 and 1:10 molar ration of vector to insert. Remember to setup negative (and if possible positive) control. Do not use more than 0.5l of T4 DNA ligase and do not use extensively frozen and thawed T4 ligase buffer (as then it would be without any ATP due to repeated freezing and thawing) Incubation temperature should not be more than 25C, if possible. (I usually leave it in 15C incubator overnight)
And honestly, I never run ligation on the gel..I know some people do it but thats not my method of choice.

-noelmathur-

Ligase binds DNA and will retard and spread gel bands when present along with the DNA. If you want to visualize ligation reaction results, you should heat kill the ligase (20 minutes at 65C) before running the gel. This works poorly for ligation reactions containing PEG such as virtually all "quick ligation" buffers. For cohesive end ligations, regular ligase works as well or better than those quick ligation buffers.

-phage434-

priyak on Mar 18 2009, 12:55 PM said:

hi
I prepared a ligation mix of 15microlitr. My insert was of 1640 bp and vector of 4000 bp. after incubating in RT for 1hr, i checked the mix in 1% agarose, butni didnt find any band except the marker...Can anyone give me a goodsuggetion.


priya

How much DNA did you load?

-swanny-

Did you transform the ligated products? If yes, did you get colonies? I usually don't run a gel to check after ligation. What I do is to transform and do a colony PCR to check if there are ligated products.

-jiajia1987-

jiajia1987 on Mar 18 2009, 06:32 PM said:

Did you transform the ligated products? If yes, did you get colonies? I usually don't run a gel to check after ligation. What I do is to transform and do a colony PCR to check if there are ligated products.


same as i do, even minute amount of ligated product (even it cannot be visualized by agarose gel) can give you sufficient positive clones, just go ahead for transoformation

-Nrelo-