TITLE: phi29 DNA polymerase protocol
AUTHORS: Elena Hilario

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isothermal amplification of a few nanograms of DNA with phi29 DNA polymerase in the presence of trehalose allows for a more stringent amplification temperature that prevents unspecific primer amplification in the water control reaction.</div><br/><br/><div class = "text-block">Spiess, A. N., Mueller, N. & Ivell, R. Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin Chem 50, 1256-1259</div></div>

[STEPS]
?. [Isothermal amplification]
Set up the following profile in a PCR machine, 1 cycle only, lid at 105ºC:95ºC 3 min → 40ºC 1 min → 38ºC 12.5 h → 65ºC 10 min → 10ºC 10 min → stop
?. [Isothermal amplification]
Prepare the 1X amplification mix and keep it on ice. Besides the DNA reaction, include a water control, and an extra reaction:0.6 M trehalose; 1X NEB phi29 Reaction Buffer, 0.8 mM dNTPs, 2 Units phi29 DNA polymeraseReagent3 reactions (1 µL)Deionezed sterile water5.4 10X phi29 DNA polymerase reaction buffer61.2 M Trehalose308 mM dNTP mix6phi29 DNA polymerase 10 U/uL0.6
Reagent3 reactions (1 µL)Deionezed sterile water5.4 10X phi29 DNA polymerase reaction buffer61.2 M Trehalose308 mM dNTP mix6phi29 DNA polymerase 10 U/uL0.6
?. [Isothermal amplification]
Prepare the annealing mix, including a control (water only):10 mM Tris-HCl, 1 mM MgCl2, pH 82 µL2 µLPT-random hexamer, 10 pmol/µL1 µL1 µLGenomic DNA ~10 ng/µL1 µL-Deionized sterile water-1 µL
10 mM Tris-HCl, 1 mM MgCl2, pH 82 µL2 µLPT-random hexamer, 10 pmol/µL1 µL1 µLGenomic DNA ~10 ng/µL1 µL-Deionized sterile water-1 µL
?. [Isothermal amplification]
Mix and incubate in PCR machine Run the 95°C and 40°C steps, pause the machine
?. [Isothermal amplification]
Add 16 μL of 1X amplification mix. Vortex the tube and quickly spin down for few seconds. Return the tube to the PCR machines and continue with the amplification profile
If concerned with such a long incubation, you can add a drop of mineral oil before resuming the PCR profile
?. [Isothermal amplification]
Spin down the tube and transfer the contents to a 1.5 mL tube. Rinse the PCR tube with 10 μL sterile deionized water and transfer it to the 1.5 mL tube. Mix briefly. Final volume ~ 30 μL
?. [Quantification and quality check]
Quantify the amount of DNA using 1 μL of the amplification reaction and the HS dsDNA Qubit kitQuantify 1 μL of the water control reaction as well
?. [Quantification and quality check]
Depending on the yield, load 50 - 100 ng of amplified DNA in one lane of a 1% agarose gel in 1X TAE. Load ALL the water control reaction in another well. Load 4 μL of the 1 Kb Plus ladder in a separate laneRun the gel at 140 V for 30 min
?. [Quantification and quality check]
When finished, transfer the gel to the staining box containing 50 mL 1X TAE buffer and 3 μL SybrSafe. Close the box and shake gently for 10 min at room temperatureTake a photo of the gel. If the bands are too fain, place the gel on a new box with tap water, close the box and shake gently for 2-4 min to destain the gel slightly and take the photo again
?. [Expected result]
The amplified sample can be trusted only if the water control is blank
Alternatively, the sample can be analyzed in either the BioAnalyzer or the Fragment Analyzer, where you only need 2-5 ng