Pcr Reaction

There are four major steps involved in a colony PCR reaction: 1) growing bacteria and preparation of whole cell suspensions; 2) making a PCR master mix and aliquoting this into Eppendorff tubes; 3) performing PCR with suitable cycling conditions; 4) analyzing amplification products on an agarose gel. In general, a reaction mixture of 25 pL contains a final concentration of 200 pM of each deoxynucleoside triphosphate (dNTP), 2.5 pL 10 x reaction buffer (100 mM Tris-HCl at pH 8.3, 500 mM KCl), 1.5 mM MgCl2, and 0.1-1.0 pM of each of the upstream and downstream primers, together with 2.5 units of Taq DNA polymerase. A portion (2.5 pL) of the whole-cell preparation of template DNA is also required. The final volume of this mix should be adjusted to 25 pL with sterile water. The PCR cycle should consist of an initial denaturation step at 95°C for 2-10 min (depending on the

Taq DNA polymerase being used), followed by 30-35 cycles of amplification with denaturation at 95°C for 30 sec, annealing at 50-60°C for 30 sec, and extension at 72°C for 30 sec, ending with a final extension at 72°C for 7 min. Part (5-10 mL) of the PCR product should be loaded onto a 0.5 x TBE-agarose gel for analysis. Products from 0.1 to 1.5 kb are easily resolved on a 1.5% gel under UV light after staining with 0.5 mg/mL of ethidium bromide solution for 30 min.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment