Protocol pgem easy pdf

The pgem rt 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. In less than an hour, starting with less than 2 mls of cell culture, this protocol. The 1218 version of this technical manual was revised to remove references to discontinued products in the notes on sequencing primers in section 5. View notes pgem from chem 1a at san jose state university. Pgemt and pgemt easy vector systems protocol molecular.

Ordering information the number of white coloniesng vector dna 400 and 2,400 bp. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube. Prepare 1 lbampicillinxgal lba plates are 1ml of 100mm. If a proofreading dna polymerase is used, a overhangs will need to be added. Transformation protocol when using the pgemt and pgemt easy vector. Subcloning by restriction digest is a commonly used lab technique. A protocol for cloning pcr products using t vectors. The protocol below is for a blunting reaction following restriction digestion and has been tested with the buffers listed above. Also, because our pdf size converter and tools are universal, you can upload and convert or compress pdf. The plasmid contains t7 and sp6 rna polymerase promoters flanking a multiple cloning region within. As long as you have an internet connection, you can access our pdf conversion tools and compress a pdf online free. How to multiply pgem t easy vector in the laboratory. We have generated a multifunctional vector, pgem wiz, to assemble inverted repeats targeting any drosophila genes by shuttling intron 2 of the drosophila gene white from pwiz into a cloning vector, pgem s1 fig. You may consider to ask the plasmid from the group to try out in your lab.

The highcopynumber pgemt and pgemt easy vectors contain t7 and sp6. Although the percentage of recombinants obtained usingthe alternative protocol was reduced, the low levels of background i. Specifies the server message block smb protocol versions 2 and 3, which support the sharing of file and print resources between machines and extend the concepts from the server message block protocol. Plasmid cloning by restriction enzyme digest aka subcloning. May i know if it is true that gene with any sequence also can be inserted into pgem t easy. You can choose to use mach1tir cells which grow quicker and the ligation only takes 30 minutes.

I have been trying to subclone an insert having 2300 bp into pgem t easy vector. Server message block smb protocol versions 2 and 3. Instructions for use of products a60, a80, a3600, a3610. Since it has atoverhang, so will this affect the type of gene. Transformation protocol when using the pgemt and pgemt easy. Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. I tried to transform the ligated vector into li dh5. Pgem t and pgem t easy vector systems protocol free download as pdf file. They offer all of the advantages of the pgemt vector systems with ecori and. In addition, the cloning process can be quicker than the pgem system. For whiteblue colony screening by pgem t easy vector a high copy number plasmid, you need to use lbamp plate supplied by iptgxgal prepare 1l of lb plates as follows. Technical manual pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. This pcr cloning kit contains an optimized 2x cloning master mix with a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low.

For whiteblue colony screening by pgemt easy vector a. Pmod anatomy tool pgem 3 the anatomy tool supports various types of analyses and simulations based on anatomical data. A versatile zero background tvector system for gene cloning and. Rna polymerase promoters flanking a multiple cloning region within the. Quick protocol 1 pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. The primers i used have snabl and notl site in forward and reverse primer end respectively. Transformation protocol when using the pgemt and pgemt. Pdf a low cost dna molecular weight marker was produced by a marker primerdirected synthetic method using pgem t easy vector as the dna template find, read and cite all the research you. Followed the ligation protocol as per promega instuctions. Here follow a detailed protocol as i usually do 1x taq buffer. Briefly centrifuge the pgemt or pgemt easy vector and control insert. Also, because our pdf size converter and tools are universal, you can upload and convert or compress pdf files from any device and operating system you might be using, from anywhere you happen to be. Ordering information the number of white coloniesng vector dna 400 and 2,400bp. For the purposes of this tutorial we will discuss how to move a cdna from one plasmid to another.

Convert multiple pdfs at once, design workflow automation, and use your current dropbox folders as input and output location. Prepare 1 lbampicillinxgal lba plates are 1ml of 100mm amp in a liter, 32 ul of 50 ugul xgal per plate. Protocol for ligations using the pgem t and pgem t easy vectors and the 2x rapid ligation buffer 3. Before setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector to use for the ligation reaction.

Overview of genomic dna library construction using pgem. Transformation protocol when using the pgem t and pgem t easy vector ligation reactions 1. Ta cloning ligation of insert with pgem easy vector. Pgemt is a linearized cloning vector that can not be multiplied.