Team:CSU Fort Collins/Experiments

COLORADO STATE
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Protocols

PCR

Piece Amplification

Component	50μl reaction
Molecular grade H₂O	Added first. 32.5μL
5x HF Buffer	10μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Template DNA	1.0μL
DNA polymerase	Added last. 0.5μL

Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	45s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

Colony PCR

Component	50μl reaction
Molecular grade H2O	Added first 33.5ul
5x Phusion HF Buffer	10μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Colony	1
Phusion DNA polymerase	Added last. 0.5μL

Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	45s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

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Digestion

Component	50μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 50ul
Custsmart Buffer(different if using pstI)	5ul
DNA	1ug
Enzyme 1	.5ul
Enzyme 2	0.5ul

Procedure

Digest your mixture in a water bath at 37C for 60min

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Ligation

Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1

Component	20μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 20ul
T4 DNA Ligase Buffer	2ul
Insert DNA
Backbone DNA
T4 DNA Ligase	1ul

Incubate at 16C overnight on a heating block or at room temperature for 1 hour.

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Transformation

Materials

Transformation (Need 2x LB+Antibiotic plates. Check for plates. Check for hockey spreaders or glass beads):
LB Media
Ligated back bone
Digested back bone(control)

Procedure

Set water bath to 42C
Remove LB+Antibiotic plates from 4C and allow them to come to RT.
Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
Add 5μL of ligation mix chemically competent cells.
Add 5uL of digested back bone to chemical competent cells as a control
Incubate on ice for 30 min.
Heat shock cells for 60 sec at 42C without shaking.
Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.
Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.

LB Media and Plates

LB Media

Material

LB Miller (Powder)
1L Glass Bottle
Stir Bar
DI/RO Water

Procedure

Triple rinse bottle with DI water
Add stir bar to the bottle
Add 700 mL of DI Water to the 1L Bottle
Add 17.5 g of LB Miller
Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
Place autoclave tape on top
Autoclave on Liq 30
Let cool and then tighten the cap and store at room temperature

LB Agar Media for Plates

Material

LB Miller (Powder)
1L Glass Bottle
Stir Bar
DI/RO Water
Agar
Antibiotic if applicable
Petri Dish Plates
Ethanol Proof Markers
Tape

Procedure

Triple rinse bottle with DI water
Add stir bar to the bottle
Add 700 mL of DI Water to the 1L Bottle
Add 17.5 g of LB Miller
Add 8.4g of agar (not agarose)
Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
Place autoclave tape on top
Autoclave on Liq 30
Remove IMMEDIATELY from autoclave when finished
Place on stir plate and slowly stir (no air bubbles from stirring too fast) until the bottle can be held comfortable for a 7 seconds (20-30 min of cool down time)
Add 700 μL of aliquoted Antibiotic
INSIDE THE HOOD:
1. Spray the sleeve of plates, the agar media, the marker, and the tape down with ethanol before putting them in the hood. When opening the sleeve of plates, be careful to not rip it because you will put the plates back into it.
2. Following sterile technique pour the plates about a 3rd of the way full
3. Allow to cool in the hood until they have solidified and are no longer warm
4. Turn the plates upside down and put back in the sleeve
5. Tape the sleeve shut and write the antibiotic and the date they were made on the tape.

Plasmid Construction

What is the template for your PCR:

Primer A:_________ Tm=__C

Primer B:_________ Tm=__C

Component	50μl reaction
Molecular grade H2O	Added first. 32.5μL 33.5ul for colony PCR
5x Phusion HF Buffer	10μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Template DNA	1.0μL 0uLcolony PCR
Phusion DNA polymerase	Added last. 0.5μL

Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	45s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

Measure concentration of clean-up PCR products with a nano drop
Run 10µL of clean-up PCR with 2ul of SybrGreen DMSO products on a gel 1%Agarose Gel (110V for 60mins). Look for How Many Base Pairs your insert is
Digest (Insert) and (Backbone )with ________+__________ at 37oC for 90mins.

Component	50μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 50ul
Custsmart Buffer(different if using pstI)	5ul
DNA	1ug
Enzyme 1	.5ul
Enzyme 2	0.5ul

Clean up all DNA fragments with PCR clean-up kit.
Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1
Incubate at 16C overnight on a heating block.

Component	20μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 20ul
T4 DNA Ligase Buffer	2ul
Insert DNA
Backbone DNA
T4 DNA Ligase	1ul

Transformation (Need 2x LB+Antibiotic plates. Check for plates. Check for hockey spreaders or glass beads):
- Set water bath to 42C
- Remove LB+Antibiotic plates from 4C and allow them to come to RT.
- Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
- Add 5μL of ligation mix chemically competent cells.
- Add 5uL of digested back bone to chemical competent cells as a control
- Incubate on ice for 30 min.
- Heat shock cells for 60 sec at 42C without shaking.
- Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.
- Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
- In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
- Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.
Next day, pick and annotate (give each colony a #) 16 colonies onto new LB+Antibiotic plates. Place new plates in 37oC overnight.
Verify all 16 colonies via PCR with Taq DNA polymerase(DO NOT USE PHUSION!!!) using these primers:

Primer A:_________ Tm=__C

Primer B:_________ Tm=__C

Component	50μl reaction
Molecular grade H2O	Added first. 38.5ul
10x Standard Taq Buffer	5μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Colony	1
Taq DNA polymerase	Added last. 0.5μL

Use the following thermalcycler protocol:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	95	5 min	1
Denaturation	95	30s	35
Annealing	LowerTm+1	30s	35
Extension	68	41min/kb	35
Final Extension	68	5min	1
Hold	4	Hold	1

Run 10µL of all 16 PCR with 2ul of SybrGreen DMSO products onto 1% Agarose gel (110V for 60mins).
Pick best two mutants and start overnight cultures in 2mL LB + Antibiotic. Leave them to grow in the 37C incubator overnight.
Next morning; miniprep them (there should be 4 plasmids). Measure their concentrations with the nanodrop.
sequencing

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Gel Electrophoresis

Materials

Agarose
1X TAE Buffer
Parafilm
Electrophoresis chamber and power source
SYBR Green
Loading Dye
DNA Ladder
PCR Samples

Procedure

Using a balance, mass out 0.5 gram of agarose (for 1% gel, for 2% mass out 1.0 gram). Mix this with 50 mL of TAE 1X Buffer.
Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).
Pour the mixture into a gel plate and insert a correctly-sized comb (8, 10, etc. lanes). Allow to cool and solidify on the benchtop.
Mix your samples on a piece of parafilm. Use 10 μL of the appropriate DNA Ladder (1 kb, 100 bp, 2 log, etc.) combined with 1 μL SYBR Green/DMSO in the first lane. For all samples, mix 10 μl of sample and 2 μL of SYBR Green/Dye Mixture.
Place gel in tray into the gel electrophoresis apparatus - wells should be close to the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill - there is a guide sticker on the apparatus which marks the max fill line.
Load 10 μL of each sample into the appropriate wells.
Connect wiring and run gel electrophoresis at 110 V for 1 hour.
Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.

Experiments

Growth on Fatty Acids

Materials

Material	Amount
Something	ul
Something	ul
Something	ul

Procedure

First
Second
Third

Terpenoid Growth Curve

Materials

Experimental strains
LB
Chloramphenicol
LB Powder
Glucose
IPTG
DI water
Shake flasks
Syringe filter
Quartz cuvettes
50 mL centrifuge tubes

Procedure

Create two different strains of E. coli to compare.
- Strain 1: DH5Α E. coli with lac promoter
- Strain 2: DH5Α E. coli with lac promoter and genes of interest
From glycerol stock, create three 2 mL overnight cultures in LB of each E. coli strain (total of six cultures. Incubate at 37 degC and 225 rpm overnight
Prepare solutions and glassware

Make 150 mL stock solution of glucose at 200 g/L
Make 225 mL stock solution of LB media (7.5 g in 225 mL)
Make 10 mL stock solution of IPTG at 1 M

Autoclave six 250 mL shake flasks, the LB, and the glucose stock solutions using a Liquid 30 cycle
Prepare 50 mL of media in shake flasks

12.5 mL of glucose solution at 200 g/L for a concentration of 50 g/L
37.5 mL of LB media at >1X for a concentration of 1X
25 uL of IPTG solution at 1 M for a concentration of 0.5 mM

For each overnight culture, take an OD₆₀₀
Use C₁V₁ = C₂V₂ to calculate the volume of overnight culture required to inoculate the culture at OD₆₀₀ of 0.05
Incubate shake flasks at 37 degC and 225 rpm
Take OD measurements

For the first six hours (starting at the first hour) take one 750 uL samples each hour
Then take one 100 uL sample every 12 hours until the total incubation time reaches 72 hours
Measure each sample in the OD reader at 600 nm; for the 100 uL samples, dilute them 1:10 with 900 uL of blank solution before measuring

At the end of the experiment, remove 1 mL of suspension from each flask and store in a microcentrifuge tube to use for HPLC analysis
Remove 25 mL of suspension from each flask and store in a 50 mL centrifuge tube

Spin down cells at 5000 x g for 15 minutes
Transfer supernatant to another 50 mL centrifuge tube
Store cells and liquid at -80 degC

Terpenoid Extraction and Detection

Materials

First
Second
Third

Procedure

First
Second
Third

KillerRed Kill Curve

Materials

First
Second
Third

Procedure

First
Second
Third