Mike Barnkob:Protocols/Cloning/PCR

From OpenWetWare

Jump to: navigation, search

Front page

Contents

PCR Reaction

How to set up an simple PCR reaction.

Protocol 1: Q5 High-fidelity Polymerase

Reagents and setup

PCR reaction mix:

  1. NEB Q5 High-Fidelity DNA Polymerase Master Mix
  2. Invitrogen 10 mM dNTP Mix

Thermal cycler setup version 1:

  1. Check Tm of primers using NEB Tm calculator and adjust step 3 below accordingly.
  2. Program thermal cycler as follows:
    • Step 1: 95°C for 3 minutes, initial denaturation
    • Step 2: 95°C for 10 sec
    • Step 3: 50-72°C for 30 sec
    • Step 4: 68°C for 60 sec pr. kilobase fragment
    • Repeat step 2-4 35 times
      Note: For longer fragments, increase to 40.
    • Step 5: 68°C for 2 min, final extension
    • Step 6: 4°C for ∞

Thermal cycler setup version 2:

  1. Check Tm of primers using NEB Tm calculator and adjust step 3 below accordingly.
  2. Program thermal cycler as follows:
    • Step 1: 98°C for 60 sec, initial denaturation
    • Step 2: 98°C for 10 sec
    • Step 3: 50-72°C for 30 sec
    • Step 4: 72°C for 20 sec pr. kilobase fragment
    • Repeat step 2-4 33 times
    • Step 5: 72°C for 2 min, final extension
    • Step 6: 4°C for ∞

Protocol

Setup reaction:

  1. Prepare primers at 10 μM concentration.
  2. Mix following x samples:
    • 1-2 μL template DNA in each tube (genomic: 1-200 ng, plasmid: 1 pg - 1 ng)
    • dH20 (up to a total of 25 μL)
    • 5 μL 5X Q5 Reaction Buffer
    • 0.5 μL dNTP mix (10 mM)
    • 1 μL Forward primer (10 μM)
    • 1 μL Reverse primer (10 μM)
    • 5 μL GC Enhancer Buffer
    • 0.25 μL Q5 High-Fidelity DNA Polymerase
  3. Load tubes/plate into thermal cycler and run.

Quality control:
Gel:

  1. Make 0.8-1% agarose gel
  2. Mix in Eppendorf tube:
    • 9 μL Blue Loading Dye (6X)
    • 1 μL DNA sample
  3. Run the gel at 100 V until the fastest dye has moved 2/3 of the gel length

Storage:

  • DNA may be stored at –20°C.

Protocol 2: MangoTag

Sarah style PCR for colony PCR.

Reagents and setup

PCR reaction mix:

  1. MangoTag
  2. MangoTag buffer
  3. MgCl2
  4. dNTPs

Thermal cycler setup:

  1. Check Tm of primers using NEB Tm calculator and adjust step 3 below accordingly.
  2. Program thermal cycler as follows:
    • Step 1: 95°C for 60 sec, initial denaturation
    • Step 2: 95°C for 20 sec
    • Step 3: 50-72°C for 20 sec
    • Step 4: 72°C for 40 sec
    • Repeat step 2-4 32 times
    • Step 5: 72°C for 7 min, final extension
    • Step 6: 4°C for ∞

Protocol

Setup reaction:

  1. Make master-mix x samples (+1):
    • 5 μL Mango Buffer
    • 2.5 μL dH20
    • 1 μL MgCL2
    • 0.5 μL dNTP
    • 0.25 μL forward primer (100μM)
    • 0.25 μL reverse primer (100μM)
    • 0.5 μL MangoTag polymerase
  2. Add 1-2 μL of DNA template / bacteria into labeled PCR tubes
  3. Add 10 μL master mix into each tube
  4. Load tubes into thermal cycler and run.

References

Personal tools