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.

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