The pgemt easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. In less than an hour, starting with less than 2 mls of cell culture, this protocol should yield sufficient. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Prepare 1 lbampicillinxgal lba plates are 1ml of 100mm amp in a liter, 32 ul of 50 ugul xgal per plate plates for each ligation reaction. Subsequent transformations were performed according to standard protocols 7. Transformation protocol when using the pgemt and pgemt. The pgemt easy vector systems are convenient systems for cloning pcr. You can choose to use mach1tir cells which grow quicker and the ligation only takes 30 minutes. To release the amirna precursor fragment, any sites of the pbsk multiple cloning site can be used, since they are part of the pcr fragment. Atailing of pcr products and cloning in to pgemt easy works reliably, sequencing can be carried out with oligonucleotides a and b. Two different strategies were used to produce tvectors, i. Transformation protocol when using the pgemt and pgemt easy vector. Spin down new, empty phase lock tubes for 1 minute. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube.
A versatile zero background tvector system for gene. T7 rna polymerase transcription initiation site 1 sp6 rna polymerase transcription initiation site 141 t7 rna polymerase promoter. This allows the insert dna to be removed with a single restriction digest using either of these enzymes. Xx cc pgemt has dt, which improves efficiency of ligation of pcr product. I find that it is more efficient than pgemt easy and that i get more white colonies with my insert. Page 4 revised 507 ggaga gctcc caacg cgttg gatgc atagc ttgag tattc tatag. They offer all of the advantages of the pgemt vector systems with ecori and. The pgemt vector systems are convenient ta vector systems for quickly. Promega has provide with a positive control insert along with the kit.
T overhangs of a linearized vector by restriction endonuclease digestion kovalic et al. Transformation protocol when using the pgemt and pgemt easy vector ligation reactions 1. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect the contents at the bottom of the tubes. Protocol for ligations using the pgem t and pgemt easy vectors and the 2x rapid ligation buffer 3. In addition, the cloning process can be quicker than the pgem system. I tried to transform the ligated vector into li dh5 alpha by electroporation but got no results. These single 3t overhangs at the insertion site greatly improve the efficiency of ligation of a pcr product into the plasmid.
Protocol for ligations using the pgemt and pgemt easy vectors and the 2x rapid ligation buffer 3. This website uses cookies to ensure you get the best experience. Rna polymerase promoters flanking a multiple cloning region within the. The pgemt vector is ready to use in ligation reactions, prepared by cutting the pgem5zf. The robertson lab protocols list perelman school of. T%20and%20pgemt%20easy%20vector%20systems%20protocol. The pgemt easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and noti, providing three singleenzyme. Transformation protocol when using the pgemt and pgemt easy. Ligation we recommend using the quick ligation kit new england biolabs for this reaction. The high copy number pgemt and pgemt easy vectors contain t7 and sp6. Department of biotechnology, iit madras, chennai 600 036, india. Mar072008 this sounds dumb and i think i know the answer but looking at the vector map has me confused. The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products.
By continuing to use this site, you agree to the use of cookies. Followed the ligation protocol as per promega instuctions. For whiteblue colony screening by pgemt easy vector a. They claimed that their created new version pgemft for ta cloning has a better performance then pgemt vector. Analysis of recombinants obtained from cloning of a 7. Quick protocol 1 pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610. Riboprobe synthesis for in situ hybridization martindale lab. Prepare 1 lbampicillinxgal lba plates are 1ml of 100mm. The only difference between pgemt and pgemt easy is in the multiple cloning site mcs. A protocol for cloning pcr products using t vectors. The toverhangs 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. Researchers have tried to develop pgemtlike tvector. A versatile zero background tvector system for gene cloning and. Briefly centrifuge the pgemt or pgemt easy vector and control insert dna tubes to collect the contents at the bottom of the tubes.
How to multiply pgem t easy vector in the laboratory. Pdf a low cost dna molecular weight marker was produced by a marker primerdirected synthetic method using pgemt easy vector as the dna template find, read and cite all the research you. There is no genbank accession number for the pgemt easy vector. Incubate at room temp for 24 hours, or at 4c overnight.
The parent vector is linearized at the position indicated by in this pgemt easy vector sequence and a t is added at each end. Pcr products can be easily cloned into a linearized vector with 3. Ordering information the number of white coloniesng vector dna 400 and 2,400 bp. Included in this guide are three protocols for cloning of gene fragments into plasmids for functional use. Ordering information the number of white coloniesng vector dna 400 and 2,400bp. The mcs of the pgemt easy vector contains sequences on either side of the insert that are recognized by the restriction enzymes not i and ecor i.
Transfer the aqueous phase to the new phase lock tubes. If initial experiments with your pcr product are suboptimal, ratio optimization may be necessary. Get cells out of freezer, thaw on ice for 5 minutes 2. Each of these protocols has been demonstrated with one or more of the e. Yes you can insert any gene sequence in pgemt easy vector but one thing you have to. Equilibrate the plates to room temperature 37 c prior to plating. Briefly centrifuge the pgemt or pgemt easy vector and control insert.
608 601 637 732 1333 1622 643 1505 1573 355 1008 566 365 891 1213 484 1057 815 1465 970 676 499 1008 1443 427 61 30 601 1460 1273