All reagents (containing vector and ligation reagents) are stored at -20°C. Aliquoting could be taken to avoid repeated freezing and thawing.

The pGM-T Vectors are linearized vectors with a single 3´-terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the efficiency of ligation of PCR products by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases.

The pGM-T Vectors are high-copy-number vectors containing T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding region of the enzyme β-galactosidase. Insertional inactivation of the α-peptide allows identification of recombinants by blue/white screening on indicator plates.

1. It's necessary to use the control fragment during the transformation process, to confirm the reasons of the problems coming out in the experiment.

2. It's suggested to leave part of ligation product. If the last step appeared problem, you could remedy quickly and don't have to repeat the ligation step.

3. The bacterium volume adding on containing antibiotic SOB or LB solid culture agar could be adjusted according to the experiment. If the quantity of transformation DNA was large, the bacterium volume could be less than 100 μl; contrarily, the bacterium volume could be 200~300 μl. If it’s estimated that the clone was less, it could centrifuge 4,000 rpm for 2 min firstly, and then take out part of culture medium. Mix the remaining medium and bacterium, then extract suitable bacterium to add on containing antibiotic SOB or LB solid culture agar. Other remaining bacterium could store at 4°C. If the transformation bacterium colonies was less in the next day, it could extract stored bacterium to add on new containing antibiotic SOB or LB solid culture agar.