Dear fellows, I am trying to insert a gene of almost 10 Kb into pCAMBIA 1305.1 plasmid (12.8 Kb) digested with respected enzymes. I have divided the gene into two fragments (S1 and S2) of 5 kb each. I have successfully inserted S1 and now the size becomes almost 17 kb. Now I am trying to introduce the remaining S2 into partially made vector but couldn't succeed after many attempts. I have tried different vector to insert ratios but couldn't get any colonies. I need your precious suggestions for the task and will be very thankful if you can please share your knowledge and experience. Thank you very much.
Cloning a large gene is undoubtedly laborious but obviously acievable. I successfully subcloned a 15kb insert into 3kb vector! Certain tips can be helpful
1. Make sure that your REs are of high fidelity class
2. After digestion, try to gel purify each fragment to obtain relatively pure products
3. Insert: vector ligation ratio of either 3:1 or 5:1 mostly work with high efficiency. Note that the ratio is Molar ratio and not mass ratio. To simply determine the molar ratio, go to NEBiocalculator/ligation on NEB website. The total mass of vector and insert of around 200 ng should be fine.
4. while it is generally recommended that 5ul of ligation mixture be used for transformation,10 ul of the ligation reaction often gives me good result.
5. Try to use commercially available competent cells and not those you prepare by yourself. This is to achieve high transformation efficiency. Remember, you are dealing with a large construct. Selection of competent cells that have minimal effect on insert stability is imperative.
6. Large constructs are better of in a low copy number plasmid. This is to minimise DNA loss during propagation in bacteria.
7. Always have controls, to determine at which stage things go crazy.lol
Finally, if you divide your 10kb insert into two fragments, make sure they are overlapping each other. For instance the first fragmant can be 1-5500nt and the second fragment 5000-10000nt, depending on the restriction map of the insert.
Hope this helps
Bashir
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