Design Primers

1. Locate Genetic Sequence of Interest
• Check: do you need to add a restriction enzyme site to the primers to aide in contruction?
Other considerations:
• Primer length should be between 18-30 base pairs (bp)
• Avoid repeats of 4 or more
• The melting temperature (T_m) of the two primers which amplify one gene should be close together and between 52 °C and 65 °C
• At the 3' end, you should end with a G or C, avoid a T or mismatches, and avoid runs of 3 or more G/Cs in the last 5 bps at the 3' end
• G/C content should be 40-50%

PCR Amplification of Gene

1. Isolate DNA to be amplified
• If DNA is located on a plasmid in E. coli, use a plasmid miniprep kit to isolate plasmid DNA (follow kit instructions) from an overnight culture of backteria
• If DNA is located in the genome of some organism, do the appropriate genomic DNA extraction
2. PCR amplification
Once you receive primers:
• Spin vials for 5 minutes at maximum speed in the microcentrifuge
• Make a 100 μM stock solution using molecular grade (nuclease-free) water
• Vortex for at least 1 minute
• Dilute stock solution 1:10 fold for a working solution of 10 μM
• Follow PCR reaction mix as described in Table 2-1



Table 2-1: PCR Reaction Mix
Component	Volume (μL)	Concentration (μM)
5X Phusion Buffer	10	1X
10 mM dNTPs	1	200
Primer A	2.5	0.5
Primer B	2.5	0.5
Template DNA	~1	-
Phusion DNA Polymerase	0.5	0.2 U/μM
Nuclease-Free Water	Fill to 50 μL	-
Total	50	-


Note: Protocol will change for different enzymes

• Use New England Biolabs' (NEB) Q5 2X Master Mix, perform at least 2 reactions so that you have plenty to work with in downstream applications
• Pipette mixture up and down
• Complete PCR Thermalcycler program as described in Table 2-2



Table 2-2: PCR Thermalcycler Program
Cycle Step	Temperature (°C)	Time	Cycles
Initial Denaturation	98	30 s	1
Denaturation Annealing Extension	98 Lowest Tm + 3 72	10 s 15 s 30 s/1 kbp	30
Final Extension	72	10 min	1
Hold	4	-	-




• Run electrophoresis gel to check PCR product
3. Use PCR clean-up kit to clean up PCR samples if successful
• Store PCR product at 4 °C or -20 °C until further use
• If PCR unsuccessful, refer to manual for further troubleshooting suggestions

Isolate the target plasmid

1. Identify if your plasmid is high-copy or low-copy
• For a high-copy plasmid, the miniprep from one 2 mL overnight culture should be sufficient
• For a low-copy plasmid, minipreps from 2 to 4 mL overnight cultures should work
2. Store plasmid at -20 °C until further use

Restriction Enzyme District

1. If cutting with two enzymes identify if there is a buffer that will give good activity for both enzymes
• If yes: proceed using that buffer
• If no: first cut with one enzyme then purify with PCR clean-up kit; then cut with 2^nd enzyme
Refer to NEB Website for help in setting up an enzyme digest
2. Cut both PCR product and backbone (plasmid) using Table 2-3 as a rough guide.



Table 2-3: Enzyme Digest Mix
Component	Volume (μL)
10X NEB Buffer	5
BSA	Add to 100 μg/mL if desired
DNA	1 μg
Restriction Enzyme	10 units
Nuclease-Free Water	Fill to 50 μL
Total	50



3. Incubate for at least 1 hour at temperature corresponding to incubation temperature for enzyme used.
4. After restriction enzyme digest, use PCR clean-up kit to remove enzyme and buffer from both the PCR product and plasmid

Dephosphorylate Restriction-Enzyme-Cut Plasmid

1. Combine components as outlined in Table 2-4



Table 2-4: Dephosphorylation Mix
Component	Volume (μL)
Molecular-Grade Water	39.5
10X NEB Buffer 3	10
Plasmid DNA	50 (volume from PCR clean-up)
Alkaline Phosphotase (CIP)	0.5
Total	~100



2. Incubate at 37 °C for at least 1 hour
3. Purify using PCR clean-up kit

Ligation

1. Determine concentration of DNA of the insert and the plasmid (nanodrop or run a gel for a relative concentration)
• In the ligation mix, make sure there is a total DNA concentration of 1 - 10 ng/μL
• Usually use a ratio of 2:1 to 6:1 of insert to backbone (plasmid)
2. Add components as in Table 2-5



Table 2-5: Ligation Mix
Component	Volume (μL)
T4 DNA Ligase Buffer	2
DNA Insert	varies
DNA Backbone (Plasmid)	varies
T4 DNA Ligase	1
Nuclease-Free Water	Fill to 20 μL
Total	20



3. Incubate at room temperature for at least 1 hour
4. Heat inactivate at 65 °C for 10 minutes and place on ice to cool
5. Store at 4 °C

Transformation

1. Thaw competent cells on ice. Leave in microcentrifuge tube. Set water bath to 43 °C and allow LB + antibiotics plates to come to room temperature
2. Add 1 - 5 μL of ligation to cells
3. Incubate on ice for 30 minutes
4. Heat shock cells for 30 seconds at 42 °C without shaking
5. Place on ice for 2 minutes
6. In hood, add 250 μL of SOC media to the tube and cap tightly
7. Spread 100 μL of a 1:10 dilution of the cells on an LB + antibiotics plate
8. Incubate overnight at 37 °C
9. Store remaining culture at 4 °C. If nothing grows or a few cells grow, you can plate the remaining the next day

Confirm Correct Construction of Plasmid

1. Prepare an appropriate amount of 15 mL cell culture tubes with 2 mL LB + antibiotic
2. Streak and prepare for incubation
• Remove an LB + antibiotic plate from 4 °C
• Label 1 to X # of colonies
• From the overnight transformation plate, touch a sterile toothpick to a single colony, streak on the LB plate of the appropriate number, and place toothpick in culture tube. Repeat with other colonies
3. Incubate at 37 °C and 225 rpm overnight
Next Day:
4. Isolate the plasmid by using the miniprep kit on the overnight cultures
• If you have a large number of plasmids, check the uncut plasmids on a 0.7% agarose gel without the gene to serve as a control
5. Check the plasmid by digesting with the appropriate restriction enzyme following the mixture given by Table 2-6



Table 2-6: Confirmation Mix
Component	Volume (μL)
10X NEB Buffer	2
BSA	Add to final concentration of 100 μg/mL if desired
Plasmid DNA	5
Restriction Enzyme	0.5
Nuclease-Free Water	Fill to 20 μL
Total	20



6. Incubate for at least 1 hour at the appropriate temperature for the enzyme
7. Run the plasmid digest on a 1% agarose gel

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