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.
14
Pagina 13
techtip 0x2
A-TAILING REACTION FOR BLUNT-ENDED PRODUCTS Set up the following reaction in a thin-walled PCR tube: • 1–4µl) purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion) ( • 2µl) 5X GoTaq® Reaction Buffer (colorless or green) ( • 2µl) 1mM dATP (0.2mM final concentration) ( • 1µl) GoTaq Flexi DNA Polymerase (5u/µl) ( • 0.6µl) 25mM MgCl2 (1.5mM final concentration) ( • uclease-free water to a final volume of 10µl N Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler. After the tailing reaction is finished, 1–2µl can be used without further cleanup for ligation into the pGEM®-T or pGEM®-T Easy Vector Systems.
techtip 0x3
TIPS FOR T-VECTOR CLONING EXPERIMENTS Although T-vector cloning experiments are simple in concept, here are a few ways to ensure best results: 1. Avoid introduction of nucleases, which may degrade the T overhangs on the vector. Use sterile, nuclease-free water in your ligation reactions. 2. Use high-efficiency competent cells (≥1 × 108cfu/μg DNA) for transformations. The ligation of fragments with a single-base overhang can be inefficient, so it is essential to use cells with a transformation efficiency of at least 1 × 108cfu/μg DNA to obtain a reasonable number of colonies. 3. Limit exposure of your PCR product to shortwave UV light to avoid formation of pyrimidine dimers. Use a glass plate between the gel and UV source. If possible, only visualize the PCR product with a long-wave UV source.
resources
MORE DETAILS ON PCR CLONING • Subcloning Notebook • -Vectors Technical T Manual • roperties of Thermostable P Polymerases • rotocols & Applications P Guide, Cloning Chapter • Protocols & Applications Guide iPhone Application • Online Catalog
GoTaq® and pGEM® are registered trademarks of Promega Corporation.
15
Pagina 15
Scoor meer met een online shop in uw uitgaves. Velen gingen u voor en publiceerden catalogussen online.
Promega Australia - Monthly online Magazine - August 2010 edition Lees publicatie 2Home