These single 3 t overhangs at the insertion site greatly improve the efficiency of ligation of a pcr product into the plasmid. Dna molecules, for example, the addition of a linear pcr product into a. A versatile zero background tvector system for gene cloning and. 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. The pgem t vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Page 4 revised 507 ggaga gctcc caacg cgttg gatgc atagc ttgag tattc tatag tgtca cctaa at. The incubation period may be extended to increase the number of colonies after transformation. Construction of a high efficiency pcr products cloning t vector. Then you run the digested dna on a gel to see the fragments. Riboprobe synthesis for in situ hybridization martindale lab. Thesis for the degree of master in molecular biosciences, major biochemistry, 60 study points. Based in cleveland, ohio, the gomedia team comes up with amazing stock footage for your graphic design projects.
Get cells out of freezer, thaw on ice for 5 minutes 2. The pgem r t easy vector has been linearized with ecorv at base 60 of this sequence indicated by an asterisk and a t added to both 3 ends. They offer all of the advantages of the pgem t vector systems with ecori and noti sites flanking the insertion site. Yes you can insert any gene sequence in pgemt easy vector but one thing you. A versatile zero background tvector system for gene. Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate.
The plasmid contains t7 and sp6 rna polymerase promoters flanking a multiple cloning region within the. Why do we always need cloning into pgem for example vector, before. Promega pgemt and pgemt easy vector systems pgem t easy. The work presented in this thesis was carried out at the department of molecular biosciences. The aim of molecular cloning is to insert the gene of interest into a plasmid vector, which is then inserted into a cell that will express the protein encoded by the gene of interest. Here you can download a freebies sample pack, so you get a taste of their design stuff. The pgem t easy vector systems are convenient systems for cloning pcr products. Spin down new, empty phase lock tubes for 1 minute. The pgem t and pgem t easy systems now allow you to perform ligation reactions in as little as one hour. I tried to transform the ligated vector into li dh5 alpha by electroporation but got no results. For example, proteintagging constructs generated by gateway cloning. Transfer the aqueous phase to the new phase lock tubes. The pgem t vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. The t overhangs at the insertion site greatly improve the efficiency of.
The pgemt easy vector systems are convenient systems for cloning pcr products. Transformation protocol when using the pgemt and pgemt. They offer all of the advantages of the pgem t vector systems with the added convenience of recognition sites for ecori and noti flanking the insertion site. Our teams are in regular contact with suppliers and distributors worldwide to manage inventory of raw materials to ensure continued availability. You take three individual samples of the plasmid and digest one sample with ecori, the second sample with hindiii, and the third sample with both ecori and hindiii. This allows the insert dna to be removed with a single restriction digest using either of these enzymes. For whiteblue colony screening by pgemt easy vector a. The pgem t easy vector systems are convenient systems to clone pcr products.
Instructions for use of products a60, a80, a3600, a3610. May i know if it is true that gene with any sequence also can be inserted into pgem t easy. The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Followed the ligation protocol as per promega instuctions. In this study the performance of the 2x rapid ligation buffer is compared with that of the previously supplied t4 dna ligase 10x buffer in both onehour and 16hour ligation reactions. Learn about the latest plasmid technologies and research tools. Most commercially available competent cells are appropriate for the plasmid, e. Reactions using this buffer may be incubated for 1 hour at room temperature. 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. The only difference between pgem t and pgem t easy is in the multiple cloning site mcs. Page 4 revised 507 ggaga gctcc caacg cgttg gatgc atagc ttgag tattc tatag.
Transformation protocol when using the pgem t and pgem t easy vector ligation reactions 1. In addition, the vector contains both the sp6 and t7 rna polymerase promoters flanking the multiple cloning. The pgem t and pgem t easy vector systems have been optimized using a 1. Researchers have tried to develop pgem t like t vector. Free vector samples by the talented design team of arsenalgomedia design studio. In addition, the cloning process can be quicker than the pgem system.
Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. The improved tvector system takes advantage of the restriction enzyme xcmi to. Rapid ligation for the pgemt and pgemt easy vector systems. Have questions about your order, deposit, or a plasmid. These polymerases often add a single deoxyadenosine, in a template independent fashion, to the 3. The pgem t and pgem t easy vectorsa,b are linearized vectors with a single 3. Since it has atoverhang, so will this affect the type of gene. Equilibrate the plates to room temperature 37 c prior to plating. Thesis for the masters degree in molecular biosciences. They claimed that their created new version pgem ft for t a cloning has a better performance then pgem t vector. Pcr cloning differs from traditional cloning in that the dna fragment of interest, and even the vector, can be amplified by the polymerase chain reaction pcr and ligated together, without the use of restriction enzymes.
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