Hello Friends,
I am trying to transform 1.6kb gene and 3kb plasmid into Dh5a competent cells. I digested gene and plasmid both with double digestion, EcoR1 and Xho1. I confirmed digestion from gel picture. After purification i did ligation with T4 ligase and buffer with gene 4ul, plasmid 2ul, T4 ligase 1ul, T4 buffer 1ul and ddH2O 2ul total 10ul at 16C temp for 12 hours. After ligation i did transformation in 100ul DH5a cell with 5ul ligation mixture by heat shock method. After 12 to 16 hours incubation i could not find any growth on plates. I used 50ug/ml Kanamycin antibiotic. I also run control transformation, which contains only plasmid. But i found no colonies growth on both of the plates along with control. No contamination, no growth. The plates were clear. I tried and checked each and every step from digestion to transformation 3 times but could not solve the issue. I am much worried where problem is. Can anyone help me where i am making mistake, which step is making this trouble for me?? I need complete guidance. Also tell me the cross checking of every step. Thank you
Hi Hafiz,
I don't have much experience yet, but I had exactly the same problem, that after transformation nothing grew on both plates (self-ligation and actual ligation with gene). After asking people around and looking for potential solutions online, I found a method that finally worked.
The trick is to perform double enzymatic digestion of your plasmid with dephosphorylation overnight, without gel purification of your vector. For this you can find a protocol here: https://tools.thermofisher.com/content/sfs/manuals/MAN0012878_FastAP_Thermo_Alkaline_Phosphatase_ef0654_UG.pdf
So in your case you take your plasmid, EcoRI, XhoI, fastAP and a buffer that maintains activity of all enzymes, in your case NeBuffer 2.1 should work well. Here is a webpage for finding optimal buffer for double digestion: https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder. You incubate the mixture in 37 overnight. Later on you only deactivate enzymes by incubating ini 65 oC for 15 min. In my case the trick was to not purify the vector by gel electrophoresis, as I was loosing a lot of material in this step.
Also, the amount of material you are using for ligation should be optimal, so to ensure that you have enough vector and insert and in good stoichiometry. Here is a web calculator for that: http://www.insilico.uni-duesseldorf.de/Lig_Input.html (so, in your case, for each 100 ng of vector you should use 160 ng of plasmid. You should measure concentrations first to calculate the volumes). And here https://www.addgene.org/plasmid-protocols/dna-ligation/ you can find what types of controls you can do, but uncut vector without ligase should be enough.
For transformation you may use all your ligation mix, the more material you have the higher chance that bacteria will take up ligated construct. 100 ng of plasmid should work well for this.
Also, if nothing works, make sure that your competent DH5a are fresh, i.e. not lost their competence. It may be necessary to prepare a fresh batch of competent cells.
Hope it helps!
https://tools.thermofisher.com/content/sfs/manuals/MAN0012878_FastAP_Thermo_Alkaline_Phosphatase_ef0654_UG.pdf
https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder
As per I informed from your information, the actual plasmid volume was too low comparing to the cell volume. if DH5alpha commercial competent cells it is ok to use small volume of cells (not more than 30uL) and plasmid in between 0.90uL to 1uL (100pg/uL concentration) but in case of ligation mixture it would be good to take considerations about the total yield after ligation.
You should start digest 1 μg of plasmid / insert as the standart protocol suggestion. For the purification of cut plasmid and insert you should make sure that every steps are correct. Or if you use the gel purification kit, make sure you wash the column enough. The remaining of EDTA from running buffer might interfere many enzymatic reactions. Finally you should add cut plasmid and cut insert at the molar ratio of 1:3 or 1:6 (plasmid:insert) then use all ligation reaction.
Here is the tool.
http://www.promega.com/a/apps/biomath/index.html?calc=pmolends
For me, I normally use 50 ng of plasmid. But the amount can be more than that. I also increased it if I found no transformants.
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