Basic Protocol For Longdistance

A quality target genomic DNA extract is essential for LD-PCR, and time should be taken to develop an extraction procedure. Standard concentrations of PCR reagents do not guarantee a successful amplification, and it is necessary to optimize all components for each LD-PCR reaction. In addition, the thermal cycling parameters and DNA template concentrations have to be evaluated, with too much DNA leading to nonspecific binding or even PCR inhibition and too little DNA leading to non-detectable amplicons, especially for low copy number targets. LD-PCR amplifications have been performed in as little as 10 pL[8] and up to 100 pL[ ] reaction volume, although 50 pL is now more regularly used because of enhanced heat transfer over 100 pL. Individual reagent concentrations may or may not vary from a normal 50-pL PCR reaction; listed below are concentrations that have been used:

1. Mg2+ [MgCl2, Mg(OAc)2] concentration: Range 1.1[4] to 3.5 mM.[5] Typical concentration 2.5 mM.

2. dNTP concentration: Range 0.2[4] to 1.0 mM (non-limiting).1-9-1 Typical concentration 0.2-0.25 mM, although 0.5 mM is regularly used.

3. Primer concentration: Range 0.12[10] to 2.0 pM.[11] Typical concentration 0.4 to 1.0 pM. Usual length 21-34 bp, with a balanced Tm of >58°C (usually 65-70°C).[7]

4. Typical PCR buffer: Tris-HCl 20-50 mM (pH 8.39.2) or 20-25 mM Tricine (pH 8.5-8.7), KOAc 85 mM, >10% glycerol, 2% DMSO.[4] Other reagents used in LD-PCR buffers include Tween 20 (0.1%), BSA (150-200 pg/mL), and (NH4)2SO4 (16 mM), which is used instead of K+ salts for certain polymerases.

5. Polymerase concentration: 2.6 U for a mix Taq and Pwo polymerases;1-12-1 1.5-2.5 U for Tth polymerase^9,13,1^ 0.9/0.02-1.75/0.02 U for a mix of rTth and Vent polymerases, respectively;1-4-1 3.3 U of Expand Long-Template DNA polymerases.[10]

6. DNA template: Genomic template DNA Range 50[9]-625 ng (125 ng/10 pL reaction).[8] Typical

Table 1 Commercially available LD-PCR polymerases and kits

Kit name

Manufacturer

Enzyme

PCR buffer

Maximum amplification

KlenTaq LA

Sigma

KlenTaq-1 DNA

10X optimized buffer supplied

Up to 10 kb

Core Kit

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.

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