Hi,

I am using pFLAG-CTC vector for cloning a DNA fragment of 8.5 kb. I have been struggling but failed to get a proper clone. Last time I got a clone but it carried an insert of less than 8.5 kb.

I do not know what is the actual Problem.

My strategy is as follows:

1. Gel purify the PCR product.

2. subjected the product to NdeI and XhoI together for the period of 6 hours. (both uses same buffer that is Cutsmart Buffer of NEB).

3. I perform the solution purification of digested product.

4. I perform the ligation in the ratio of 1:1.

5. Transform the DH10B cells of E.coli. and plate in LB agar (Ampicillin, 100ug/ml).

6. Incubate it in 37 degree celsius.