Home resources plasmid files basic cloning vectors pgemt easy. The t overhangs at the insertion site greatly improve the efficiency of. May i know if it is true that gene with any sequence also can be inserted into pgemt easy. T overhangs at the insertion site greatly improve the efficiency of ligation of a pcr product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for ligation of pcr products with a overhangs. Briefly centrifuge the pgemt or pgemt easy vector and control insert. Since it has atoverhang, so will this affect the type of gene. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases.
A versatile zero background tvector system for gene cloning and. Constructing genomic libraries using the pgemt vector. The primers i used have snabl and notl site in forward and reverse primer end respectively. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. T%20and%20pgemt%20easy%20vector%20systems%20protocol. The pgemt vector systems are convenient systems to clone pcr products. The pgemt and pgemt easy vector systemsa,b facilitate the cloning of pcrc products by providing linear vectors that have a single thymidine extension. The insertion site is flanked by bstzi, ecori, and noti sites. The method is based on ultrasonic cleavage of genomic dna, blunting of the fragment ends with mung bean nuclease and addition of a single 3. Cloning efficiency was calculated as the number of white coloniesng vector dna from a. The pgemt easy vector systems are convenient systems for cloning pcr.
The pgemt easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and. The pgem t vector cloning region is flanked by recognition sites for the enzyme bstzi. Alternatively, a doubledigestion may be used to release the insert from either vector. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. A protocol for cloning pcr products using t vectors.