互联网2013-11-13
1. Program the thermal cycler as follows (note that the annealing temperature will vary depending on the Tm of your primers):
94°C 2 minutes
10 cycles of:
94°C 10 seconds
Primer Tm –5°C 30 seconds
68°C 1 minute/Kb
20–30 cycles of:
68°C 1 minute/Kb ( 20 sec/cycle)
Final extension:
72°C 5 minutes
Hold at 4°C
2. Prepare a master mix as follows. Amounts below are for a 20-μl reaction. Scale the reaction volume and multiply by number of reactions as needed.
Table 1
Component
Volume
Final Concentration
SequalPrep™ 10X Reaction Buffer
2 μl
1X
DMSO
0.4 μl
--
SequalPrep™ 10X Enhancer A or B
1–2 μundefined
0.5X–1X
SequalPrep™ Long Polymerase, 5 U/μl
0.36 μl
1.8 U
DNase-free water
to 18 μl
—
3. Pipette 18 μl of master mix into each PCR tube/well, and add:
Primer mix (10 μM each) 1 μl 0.5 μM each
Template DNA (1–100 ng/μl) 1 μl as required
4. Cap the tube/well, tap gently to mix, and centrifuge briefly.
5. Place the tube in the thermal cycler and run the program from Step 1. After cycling, samples can be stored at –20°C until use.
6. Analyze the amplification products by agarose gel electrophoresis. We recommend using E-Gel® 0.8% or 1.0% gels and the TrackIt™ 1 kb Plus DNA Ladder or 1 Kb DNA Extension Ladder (see Additional Products in ordering table).
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