PCR

Ben Carter, 9/24/2014

Jiaxin Long, Modified on 02/28/2020

This protocol uses Apex Taq DNA polymerase (Genesee Cat# 42-803B1).

''Note: Ethidium bromide is a possible carcinogen but has not been shown to be carcinogenic in animals. Handle with caution.''

Primer Stock Preparation

 * 1) Remove primer stock from the primer library. OR Briefly centrifuge unopened lyophilized primers in the blue-capped IDT vials on a tabletop microcentrifuge. Add 10µL of nuclease-free water for each nanomole of primer listed on the IDT information form. (Ex.: 87nmol = 870µL of water.) Vortex to mix.
 * 2) Make a working stock of the primers by pipetting 10µL of primer solution into a microfuge tube and diluting with 90µL of nuclease-free water. Vortex to mix. Replace blue screw-cap vials in the primer library.

Reaction Mix Preparation
'Notes: The polymerase must be added last to the master mix. Mix the master mix well by pipetting before and after addition of the polymerase. Keep the polymerase on ice and do not vortex it.'

Obtain a PCR tube strip or individual PCR tubes. If performing multiple reactions, it is helpful to prepare a master mix containing the common reagents needed for all reactions. Make enough master mix for an additional reaction to ensure adequate volume. Each reaction well needs the following components:


 * 20ng is appropriate for eukaryotic genomic DNA. If amplifying from a plasmid template, use 1ng of DNA.

Thermal Cycling
''Note: Larger amplicons require longer extension times (the 72°C step). The polymerase extension rate is 1kb/min. Primers with unusual annealing temperatures may require modifications to the listed program.'' Tm Calculator: https://tmcalculator.neb.com/#!/main
 * 1) Mix each reaction tube by pipetting.
 * 2) Place the PCR tubes/strip in a thermal cycler. Run the following program:

Setting: Taq Polymerase, Taq DNA Polymerase with standard Taq buffer

Optional: Add a 4°C forever step to keep the samples refrigerated after the program is complete.

Gel Electrophoresis
Note: Amplicons smaller than ~300bp require a higher-concentration gel.
 * 1) Remove samples from the thermal cycler once the program has finished.
 * 2) Add DNA loading dye to 1x concentration. Mix by pipetting until the color is even.
 * 3) Prepare a 1% agarose gel in 1x TAE Buffer. For our electrophoresis apparatuses, we use 400mg of agarose in 40mL of 1x TAE Buffer.
 * 4) Pour TAE Buffer into the electrophoresis apparatus until it covers the gel.
 * 5) Pipette 6µL of the 1kb DNA ladder solution or equivalent into desired wells. Load the sample wells with 20µL of the samples. If overflow occurs, reduce the volume added.
 * 6) Attach electrodes (DNA runs from negative to positive). Run the gel for 60min at 100V. (Voltage and duration will vary with different apparatuses.) Higher percentage gels require longer run times.
 * 7) Visualize the DNA bands by rocking the gel in Ethidium Bromide Staining Solution for 10-15min.

50X TAE Buffer

 * 242g of Tris base
 * 57.1mL of acetic acid
 * 100mL of 500mM EDTA pH 8.0
 * Dilute to 1L with Milli-Q water.

Ethidium Bromide Staining Solution

 * 100mL of 1x TAE Buffer
 * 5µL of ethidium bromide