LIGATION DOESNT WORK - (Jan/17/2008 )

HELLO EVRYONE
whenever i set up a ligation of my vector and insert..i fail to get any colnies....i have tried different concn of insert and vector..but all in vain.
it has been quite many times now...
i am sure the ligase and the buffer are fine coz it works 4 others.
wht is the ideal volume for setting up the ligation?
whch conversion formula should one use to calculate molar ratio?

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You need to tell us a lot more. Do you have confidence that transformation is working well? How are your DNA fragments prepared? This is the usual problem, not the ligation itself. I use a 10 ul final ligation volume, and transform 1 ul.

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i have a 4.9 kb fragment whch i have to clone it in 4.7 kb GFP vector.i do a sequential digestion of my plasmid with BSTX1 and xho first eluting and finally digesting it with sac1..to get my 4.9kb fragment.it involves two elution steps.and i use qiagen gel extraction kit.while eluting my fragment i mostly elute it in 20 -30 microli od DDW b4 setting the ligation.
i normally set up my ligation in 20microlit volume and use the entire mix for transformation.
i also tried CIPing my vector bt it did not help much.

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We use 1-2 ul of a ligation reaction for transformation. Do you know how much DNA is in your ligation reaction?

There is no need for CIP as this is not going to help you in this case.

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Just a couple of suggestions to take it or leave it.

Have you tried running a portion of your ligation reaction on a gel to confirm that you have a much higher molecular weight ligated product?




If you do, then you have a problem with your transformation. In this case, you can do a positive control and transform (a very small amount 100 ng or less) some undigested GFP expression vector (I think is the vector you were ligating into) to confirm that your cells are competent and that your plates are the correct selection (Amp or Kanamycin (sp?))

If your ligations are not working, as in you do not have a ligated MW product, then more information about the ligation will be required.

Best of Luck

BeaverAggie

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i burst out in laugh to see your topic title, as you are very new here, you dont know..........i was suffering from the same problem now you are facing, and i changed my name in this forum as"ligation doesnt works"...............

chekc out your vectors, plates, antibiotic, insert fragments...........

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I have had similar problems with ligation. Now I only use homologous recombination and I swear by it. It's so much faster and easier and the best part about it is that I get results. I think that's always the most important part. All you have to do is establish homology between your vector and your gene insert (18bps is recommended). There are a few cloning kits out there, but I use one by BPS Bioscience, mainly because other ones require you to insert the gene into a shuttle vector (gateway, topo, etc.) before you can put it into your desired vector. The kit I use now lets me go from PCR product straight into my desired vector.

Hope this helps! Let me know what your results are.

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I have started using SLIC cloning. There is a commercial kit, but you can also go straight from the original paper, [attachment=4161:Li_and_E...LIC_2007.pdf]. T4 DNA polymerase is available from NEB, and RecA is through Epicentre.

One trick I have tried is to take 1ul and do a PCR using the sequencing primers for the plasmid. The most common problem with ligations would have to be incomplete digestion of the vector, so when you add the ligase, all you do is religate the vector. A PCR will tell you if that has happened. In your case, remember to give the reaction enough time to amplify the whole insert!!

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