Mike Barnkob:Protocols/Cloning/PCR: Difference between revisions
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# Prepare primers at 10 μM concentration. | # Prepare primers at 10 μM concentration. | ||
# Mix following x samples: | # Mix following x samples: | ||
#* 1-2 μL template DNA in each tube ( | #* 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) | #* dH20 (up to a total of 25 μL) | ||
#* 5 μL 5X Q5 Reaction Buffer | #* 5 μL 5X Q5 Reaction Buffer |
Latest revision as of 01:54, 1 April 2015
PCR Reaction
How to set up an simple PCR reaction.
Protocol 1: Q5 High-fidelity Polymerase
Reagents and setup
PCR reaction mix:
- NEB Q5 High-Fidelity DNA Polymerase Master Mix
- Invitrogen 10 mM dNTP Mix
Thermal cycler setup version 1:
- Check Tm of primers using NEB Tm calculator and adjust step 3 below accordingly.
- 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:
- Check Tm of primers using NEB Tm calculator and adjust step 3 below accordingly.
- 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:
- Prepare primers at 10 μM concentration.
- 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
- Load tubes/plate into thermal cycler and run.
Quality control:
Gel:
- Make 0.8-1% agarose gel
- Mix in Eppendorf tube:
- 9 μL Blue Loading Dye (6X)
- 1 μL DNA sample
- 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:
- MangoTag
- MangoTag buffer
- MgCl2
- dNTPs
Thermal cycler setup:
- Check Tm of primers using NEB Tm calculator and adjust step 3 below accordingly.
- 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:
- 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
- Add 1-2 μL of DNA template / bacteria into labeled PCR tubes
- Add 10 μL master mix into each tube
- 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/