Pgem T Easy Vector Sequencing Primer
The pgem t vector has teen linearized with ecor v at base si of this sequence indicatal by an asterisk and a t added to both 3 ends the added t is not included in this sequence the sequence shown comesponds to rna synthesized by t7 rna polymerase and is complementary to rna synthesized by sp6 rna polymerase.
Pgem t easy vector sequencing primer. 5 sequencing 1 primer. The mcs of the pgem t easy vector contains sequences on either side of the insert that are recognized by the restriction enzymes not i and ecor i. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube. T7 sp6 m13fwd or m13rev.
The incubation period may be extended to increase the number of colonies after transformation. To use taq dna polymerase for gene amplification. To see this sequence with restriction sites features and translations please download snapgene or the free snapgene viewer. Quick protocol 1 pgem t and pgem t easy vector systems instructions for use of products a1360 a1380 a3600 and a3610.
The vectors are prepared by cutting the pgem 5zf and pgem t easy vectors respectively with ecorv and adding a 3 terminal thymidine to both ends. T overhangs for easy pcr cloning. The pgem t and pgem t easy vector systemsabare convenient systems for the cloning of pcr products. My vector was pgem t easy.
The only difference between pgem t and pgem t easy is in the multiple cloning site mcs. The pgem t and pgem t easy 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. Pgem t easy parental vector for ta cloning of pcr products.
Miscellaneous sequencing primers pinpoint sequencing primer 5 dcgtgacgcggtgcagggcg 3 v4211 66 pinpoint xa 1 2 3. Becuase taq is able to add a to 3 overhang of pcr product. The insertion site is flanked by bstzi ecori and noti sites. The pgem t easy pre linearized vector contains 3 t overhangs at the insertion site to provide a compatible overhang for pcr products.
For pgem t easy vector you can use simple primer for cloning and you must. Pinpoint xa 1 t vector ptarget sequencing primer dttacgccaagttatttaggtgaca 3 q4461 55 ptarget vector melting temperatures t m.
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