Protocols · Benchling

Materials

Labware
- Thin walled 200µL PCR tubes
- Racks capable of holding PCR and microcentrifuge tubes
- Sterile pipette tips
- Permanent Marker

Equipment
- Pipettes for pipetting volume ranges from 0.25 μL to 50 μL
- Microcentrifuge capable of holding 1.5mL tubes and producing at least 17,900 x g
- Thermocycler
Reagents
- PCR Reagents as described below in Table 2.

Important information to be documented throughout this procedure

Information	Check
DNA plasmid template used in PCR reaction and concentration of sample
Expected fragment size
Primers and DNA polymerase used in the reaction
Volumes used in PCR reaction
Controls used in PCR reaction
Thermocycler used
Amplification program used
Samples’ names, dates and storage location
Unexpected and expected observations and results (either in writing or in picture form)
Deviations/Alterations of the protocol
Clarification notes (issues, significant notes)

Information

Check

DNA plasmid template used in PCR reaction and concentration of sample

Expected fragment size

Primers and DNA polymerase used in the reaction

Volumes used in PCR reaction

Controls used in PCR reaction

Thermocycler used

Amplification program used

Samples’ names, dates and storage location

Unexpected and expected observations and results (either in writing or in picture form)

Deviations/Alterations of the protocol

Clarification notes (issues, significant notes)

PCR with Phusion Polymerase

Start by filling in the table of reagents that will be added to the reaction mixture as below:

Reagent	Location	Concentration of stock solutions	Volume	Final concentration
Sterile water	-20 °C freezer		To 20 µL or 50 µL
PCR buffer: Phusion HF buffer	-20 °C freezer	5X		1X
dNTPs	-20 °C freezer	10 mM		200 μM
MgCl2	-20 °C freezer	In buffer		1.5 mM
Forward primer: ______	-20 °C freezer	10 μM		0.5 µM
Reverse primer: _____	-20 °C freezer	10 μM		0.5 µM
Template DNA: _____	-20 °C freezer	Variable		10 pg - 250 ng
Phusion DNA polymerase	-20 °C freezer	5 Units/μl*		1 unit/50uL rxn✝
Final volume			25 µL or 50uL
✝Final concentration of polymerase may vary between manufacturers	✝Final concentration of polymerase may vary between manufacturers	✝Final concentration of polymerase may vary between manufacturers	*Units may vary between manufacturers	*Units may vary between manufacturers

Reagent

Location

Concentration of stock solutions

Volume

Final concentration

Sterile water

-20 °C freezer

To 20 µL or 50 µL

PCR buffer: Phusion HF buffer

-20 °C freezer

dNTPs

-20 °C freezer

10 mM

200 μM

MgCl2

-20 °C freezer

In buffer

1.5 mM

Forward primer: ______

-20 °C freezer

10 μM

0.5 µM

Reverse primer: _____

-20 °C freezer

10 μM

0.5 µM

Template DNA: _____

-20 °C freezer

Variable

10 pg - 250 ng

Phusion DNA polymerase

-20 °C freezer

5 Units/μl*

1 unit/50uL rxn✝

Final volume

25 µL or 50uL

✝Final concentration of polymerase may vary between manufacturers

*Units may vary between manufacturers

Arrange all reagents needed in a cold block (except Taq DNA polymerase) and let them thaw completely before setting up a reaction. Gently vortex and briefly centrifuge all solutions after thawing.

CRITICALKeep the reagents on ice throughout the experiment.

Label the required PCR tubes with permanent marker and include sample details or code, as in the example of table 2 above.

Pipet the reagents following the order in table 2:

Note: MgCl₂ may be already in the buffer, verify the required final concentration for your experiment.

Note: Be careful to avoid contamination of your reagents, and DNA sources. When working with DNA and enzymes, it is highly recommended to not reuse tips.

In a separate PCR tube add all the reagents with the exception of template DNA for a negative control (increase the water to compensate for the missing volume).

When needed in your experiment, prepare in the same manner, a positive control reaction with template of known size and appropriate primers.

Gently mix the solution by “finger vortexing” (flick with finger several times), to allow dispersal of Taq DNA polymerase in the reaction mix.

Note: Care should be taken to avoid the formation of bubbles.

Briefly spin down the PCR tubes (2-3 s) using a tabletop microcentrifuge in order to ensure that all of the reagents are in the reaction mixture

Place the PCR tubes into the selected thermocycler.

Once the lid to the thermocycler is properly closed, start the required amplification program, as in table 3.

Cycle step	Cycling temperature	Time	Number of cycles
Lid Temperature	2°C higher than highest temperature	2°C higher than highest temperature	2°C higher than highest temperature
Initial denaturation	98 °C	30 s	1
Denaturation	98 °C	10 s	30
Annealing	45-72 °C	30 s	30
Extension	72 °C	30 s*	30
Final Extension	72 °C	5 min	1
Hold	4 °C	∞	1
Note: protocol may differ if using different DNA polymerases.	Note: protocol may differ if using different DNA polymerases.	Note: protocol may differ if using different DNA polymerases.	Note: protocol may differ if using different DNA polymerases.
Phusion: Follow NEB's Rules for annealing temp. Use the Tm calculator.	Phusion: Follow NEB's Rules for annealing temp. Use the Tm calculator.	Phusion: Follow NEB's Rules for annealing temp. Use the Tm calculator.

If the amplified DNA is NOT to be immediately used, label the tubes with your initials and date and temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box.

Properly record the storage location in the corresponding section in Benchling.