centrifugation as they flow through the spin column into the collection tube. Immobilized trypsin does not pass through the spin column frit, so only your peptides elute from the column. The purified peptides can be analyzed via mass spectrometry to identify the protein or assess any protein modification. Immobilized Trypsin enables: • ast Digestions: Complete F in 30 minutes. • calable Protocols: Adjusts S to protein concentration. • asy Setup: No shaking E or water baths required.
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techtips
T-Vector Cloning and a Quick Method for A-Tailing PCR Products
WITH GOTAQ® DNA POLYMERASE
Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3´ end of amplified DNA fragments. These polymerases usually add an adenine (A), leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products.
techtip 0x1
T-VECTOR CLONING The easiest method of choice for cloning PCR products is T-vector cloning. Promega’s T-vector products are pGEM®-T and pGEM®-T Easy Vector Systems. This method takes advantage of the A overhangs on the PCR product. T-vectors are linearized plasmids that have been treated to add 3´ thymidine (T) overhangs to match the A overhangs of the amplicon. The PCR fragment is directly ligated to the T-tailed plasmid vector with no need for further enzymatic treatment other than the action of T4 DNA ligase. Proofreading polymerases like Pfu and Tli do not add A overhangs, and PCR products generated with these polymerases are bluntended. Here is a simple method for adding an A-tail to DNA fragments generated by these polymerases to enable T-vector cloning.
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Promega Australia - Monthly online Magazine - August 2010 edition Lees publicatie 2Home