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DNA Ligation Not Working Troubleshooting

DNA Ligation Not Working Troubleshooting - DNA Techniques

DNA Ligation Not Working Troubleshooting - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Old 08-27-2007, 11:30 AM
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Question DNA Ligation Not Working Troubleshooting



Hi all,
My ligation is not working.

I am trying to subclone an insert from one construct to another plasmid.

Using T4 NEB and used different ratios and ligation incubation times. I have also tried highly competent bacteria to no avail.

please help me!

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Old 09-17-2007, 07:14 AM
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Default Re: DNA Ligation Not Working Troubleshooting

Hello there,
I tend to do my ligations with less DNA as it may be toxic to bacteria:
Try using 50ng vector and ~3-fold molar excess of insert.

I am almost always doing 3-way ligations which is why I use so much DNA. I think you should be able to get away with less but these amounts work reliably for me.

Try this formula with 50ng vector:



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