Lab Resources

PCR Methods

"Standard" Three Step PCR Conditions for Plasmids Templates:
         FINAL Concentrations in a 50 µl reaction:
            40-100 ng plasmid DNA per 50 µl reaction (20 to 50 fmoles)
            200 µM each dNTP
            100 ng/50 ml reaction for each primer (300 nM)
            2.5 Units Pfu Turbo Hot-Start (Stratagene)

            Cycling Parameters:
           
                        1x:     95 °C 5 minutes

                                    30x:   95 °C 30 seconds
                                               52 °C 30 seconds (or 5 degrees below Tm calculated without divalent ion effect or use AnnHyb4 program)
                                               72 °C (1 minute per kbp of expected amplicon size or minimum 60 seconds).

                        1x    72 °C 5 minutes ("polishing step").

                        1x:    4 °C "infinity"  ("hold" until manually stopped)

Typical reaction
            Stock Solutions:
            pDNA = 40-100 µg /ml
            dNTPs = 10 mM  (Promega)
            primers each = 100 ng / ml (from IDT in water at 100 ng/µl)

                                       1 x                                    2.1 rx
                                                                                 "MIX"
H2O                                    40 ml                                    84    µl
10x Pfu Buffer                      5 µl                                    10.5 µl
10 mM dNTPs                      1 µl                                      2.1 µl
pDNA                                    1 µl                                      0 µl
forward primer                     1 µl                                      2.1 µl
reverse primer                     1 µl                                      2.1 µl
Pfu enzyme                          1 µl                                      2.1 µl  (Pfu Turbo Hot Start 2.5 Units/µl)
                                            50 µl                                    102.9/2.1 = 49 µl/tube

Tube #1  NTC control               49 µl of MIX  + 1 µl water
Tube #2  PCR reaction            49 µl of MIX  + 1 µl pDNA

Protocol contributed by Dr. John Brandis.

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