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    SequalPrep Long PCR Kit with dNTPs

    互联网

    979

    实验步骤

     

    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:

    94°C                                10 seconds

    Primer Tm –5°C                       30 seconds

    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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