I am ligating an insert with a repetitive sequence and have been unsuccessful with my ligations using T4 ligase. I have a feeling that my insert is forming secondary structures that are preventing ligation. Has anybody attempted using betaine in ligation reactions?
Although I have not used betaine in ligation but intuitively, I'd be very reluctant to try strong amines or any compounds that may interact with the enzyme itself or cause a change in pH.
You may want to use bacterial strains such as Stable 3 or NEB Stbl that are chosen for their ability to do better with DNA inserts with repeats and other features causing instability.
I would definitely steer clear of altering reaction conditions that may inhibit enzyme function. Thermofishers One Shot Stbl3 or NEB Stable are what I usually use for cloning of repetitive sequences. Of the two I prefer Thermofishers One Shot Stbl3. Although they may seem expensive, you can halve the reaction volume to 25ul of cells per transformation and double the use of the kit.
Be sure to have clean insert if you have gel purified, and when using T4 ligase slow and steady usually wins the race. 16degrees overnight works well for me. Dilute the ligation reaction 1 in 2 with dH2O before transformation too! Good luck!
I am a little bit confused. If the ligation reaction itself is the problem (not sure for certain though) then how does changing the competent cell type fix the problem?
I am facing this issue with two inserts that I am attempting to subclone. One insert (4.8 kb) contains both mOrange and GFP sequences which are similar. The other insert (66 bp) is a GGGGSx4 linker sequence. In both cases I am attempting to ligate into pcdna3.0 and have had no luck. All of my other ligation reactions have been working so I feel that the issue is with the inserts themselves.
Thanks,
Certain bacterial strains are better at propagating plasmids with repetitive sequences such as lentiviral or retroviral plasmids. They also reduce the likelihood of unwanted recombination events due to the repetitive nature of the DNA. The strains mentioned above should serve you well considering you're working with repetitive sequences.
With regards to your ligation, if you can avoid blunt-end ligation I would do so. Alternatively, if you want to determine whether your ligation has worked you should design primers flanking your inserts corresponding to your destination vector, and use some of your ligation reaction in a PCR. If the ligation has worked, you will obtain a product corresponding more or less to the size of your inserts.
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