A Question about purification in making clones - (Jan/05/2005 )
Could any one tell me how many times gel purification I should perform in making clones. For EXample, I am doing a blunt-end ligation. I cut my insert from a plasmid with differet REs,and blunt the end with klneow. As to the vector ,I first cut it ,then blut the end and dephosphorylate the end. After this, I ligate the vector and insert together. So during the procedure, should I do gel purification after each step before next step?? You know, I found gel purification make lot of insert and vector lost which makes me feel upset.
Could you give me any suggestion? Thank you!!!
After every step you can try cleaning the vector by spin-collumn purification (like a PCR kit, if vector is not too large) followed by (if necessary to increase concentration) ethanol precipitation.
the first step of preparing your vector (the restriction) it is usually a good idea to purify the cut form from gel, because if you have even a small amount of uncut vector left over, this amount could give you a whole lot of background when doing the transformation.
you can test for this by a doing a control transformation with your cut vector (without gel purification) - if you get very few transformants, it should be o.k.
Thank you for your advice. I will definitely take it!