I have a problem in my cloning procedure.
Restriction enzymes (NdeI, NotI)
Vector (5.5 Kb)
Insert (0.9 kb)
After digestion I perform gel extraction for vector and insert. Then I do ligation in volume 10 micro liter, 1:3 molar ratio at 16 degrees for 16 h. but I don’t have colony on LB+Amp plates. The positive control plate is OK. I examine different molar ratio of vector and insert in ligation, also alter the T4 enzyme and Buffer, but unfortunately my problem is not solved.
Does anyone know where I might be going wrong? Are my ligation conditions ok?
Hello Azam,The Three problematic steps would be,
1. Restriction step: Did Your restriction enzyme working fine? Could you see the band of the proper size on gel after digestion?
2. I think ligation condition is ok, but if your buffer is really old, Ligation wouldn't happen properly, since ATP is the important cofactor the enzyme. So, you can add 5mM ATP in your ligation reaction or use fresh buffer which is supplied with ATP.
3. Transformation step: Even if everything is working fine, you might have been stopped here in this step. Use 5 to 10 µL of ligation mix to transform into 100µL chemically competent cells or less if you are using electro competent cells. But you said positive control plate is ok, but, there is difference between pure plasmid and ligation mixture which contains several salts and other components which affect transformation.Hence, efficiency of your comp cells should be higher.
Or, use higher concentration of vector and insert (as you will be loosing in each step),so that you will have still sufficient DNA sample for ligation.
which buffer you are using for gel analysis, TBE or TAE? If you are using TBE for elution purpose it will inhibit ligation.
OK Then no problem with the buffer.. Which company's DNA ligase and ligase buffer are you using?? check whether it requires heat inactivation after digestion..
Nice reply by Shiv Kumar. I would like to say, please check the concentration of your insert and vector. It's very crucial. You are using ligation mixture in the correct ratio provided the concentration of digested vector(at least 100ng/ul for safe side) and digested insert (at least 50ng/ul for safe side) should be appropriate. Otherwise you will have to raise the reaction volume up to 20ul. There is a loss of at least 20% DNA after every gel extraction process. So concentration matters a lot.
which kind of transformation do you use, electroporation or heat-shock?
which strain of E.Coli do you use for competents?
how old are your competent cells?
I use of heat-shock method for transformation, DH5 alpha strain and my competent cell is fresh.
D
Is your gene AT rich or GC. Please check the sequence. It must be a ligation problem. Try with quick ligation kits, sometimes they give better results.
DH5 alpha doesn't like very much "big" plasmid. Did you try to use Top10 or Stable 3?
How much ligation did you transform in competent cells? Try to increase up to 5 ul the dna you transform.
My ligation volume is 10 micro liter usually, thus I transform total of 10 micro liter.
Thank you very much for all your suggestions. Finally I could solve this problem with extension of PCR digestion time of 3 h to 4-4.5 h, It was OK and everything worked well.
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