Mike Barnkob:Protocols/Cloning/PCR

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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

• NEB, https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491
• NEB Tm calculator: http://tmcalculator.neb.com
• Qiagen's PCR Guide: http://www.qiagen.com/gb/resources/molecular-biology-methods/pcr/