Team:USMA-West Point/Experiments

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Plasmid Purification

5mL LB cultures were inoculated and grown overnight at 37C and shaking at 220rpm

Cultures were spun at 3700rcf for 5min, and supernatant was discarded

Plasmids were isolated using the DNA Plasmid Miniprep Kit (Qiagen)



25uL 2X PCR Taq Plus Mastermix (ABM Bioscience)

1uL template plasmid

1uL forward primer (final concentration 0.5uM)

1uL reverse primer (final concentration 0.5uM)

22uL water


Reactions were run on BioRad MyCycler thermocycler with the following protocol:

Step 1: 95C for 1min

Step 2: 95C for 30sec

Step 3: 52C for 30sec

Step 4: 71C for 30sec

Repeat steps 2 through 4 for 40 cycles

Step 5: 71C for 2min

Step 6: hold at 4C


The generation of PCR products was confirmed using agarose gel electrophoresis


PCR reaction clean up

PCR reactions were purified using the PCR Clean-up Kit (Qiagen) following the manufacturer’s instructions


Restriction Digest Reactions

2uL 10X reaction digest buffer (New England BioLabs)

1uL enzyme 1 (New England BioLabs)

1uL enzyme 2 (New England BioLabs)

1-10uL Plasmid or PCR insert

Adjust with water so that final volume is 20uL


All reactions were run overnight at 37C in a BioRad MyCycler thermocycler

Prior to reaction clean up, plasmids were treated with 1uL Antarctic Alkaline Phosphatase (New England BioLabs) for 1hr at 37C

All reactions were cleaned up with PCR Clean-up Kit (Qiagen) following the manufacturer’s instructions


Ligation Reactions

2uL 10X T4 Ligase Buffer (New England BioLabs)

0.5uL T4 DNA Ligase (New England BioLabs)

1-5uL of digested target plasmid (200ng of plasmid)

5-12.5uL of digested insert (600ng of insert)

Adjust volume with water to 20uL


Ligation reactions were incubated overnight at 16C a BioRad MyCycler thermocycler


Bacterial Transformations

Competent DH5alpha cells or BL21(DE3) cells (New England BioLabs) were thawed on ice

5uL of ligation reaction was added to cells and stirred

Cells/ligation solution was incubated on ice for 20min

Cells/ligation solution was placed in 37C heat block for 30sec

Cells/ligation solution were placed back on ice for an additional 2min

Cells/ligation solution were transferred to 100uL of pre-warmed SOC media

Cells in SOC media were incubated at 37C with shaking at 220rpm for 1hr

Cells were then plated on LB/Agar plates containing the appropriate antibiotic

Plates were placed in a 37C bacterial incubator at 37C overnight

Plates were removed in the morning


Colony screening

Individual colonies were used to inoculate 5mL LB cultures

Cultures were grown overnight

Plasmids were isolated using the DNA Plasmid Miniprep Kit (Qiagen) following the manufacturer’s protocol

Plasmids were then digested with restriction enzyme reactions at 37C overnight in 16C a BioRad MyCycler thermocycler

Reaction products were analyzed by gel electrophoresis



Agarose Gel Electrophoresis

0.8g of Agarose (Sigma) was dissolved in 100mL of 1X TAE (Sigma)

Agarose solution was microwaved until boiling

Molten agarose solution was used to cast gel

0.5uL of ethidium bromide stock solution was added to the molten agarose and stirred thoroughly

Gel comb was placed in mold

Gel was allowed to solidify at room temperature before being transferred to gel running box

1X TAE running buffer was added until gel was submerged

Sample prep


5uL of PCR reactions were loaded straight into gels (PCR buffer is designed for straight-to-gel analysis)

For all other samples, 4uL of 6X loading buffer (New England BioLabs) was added per 20uL volume


1uL 100bp or 1kb ladder (New England BioLabs)

2uL 6X loading buffer (New England BioLabs)

9uL water


Samples were loaded into respective wells

Gel was run at ~100V for ~40min

Bands on gel were visualized using a transilluminator (UVP)


Expression of OR1D2 and Synthetic Operon Proteins

Overnight cultures in minimal media using glycerol as the carbon source were inoculated with BL21(DE3) bacteria co-transformed with hOR1D2 and synthetic operon expression plasmids. Overnight cultures were incubated at 37C with shaking at 220rpm. The following morning, overnight culture was used to inoculate 4 fresh cultures in 20mL minimal media to an OD600 of 0.2. Cultures were grown incubated at 37C with shaking at 220rpm until the OD600 reached 0.6, when cultures were pelleted by centrifugation (3500xg for 5min), resuspended in PBS and then pelleted again by centrifugation (3500xg for 5min), before being resuspended with minimal containing either arabinose with IPTG (media had no glycerol), arabinose only (media had no glycerol), IPTG (media contained glycerol) only, or water (media contained glycerol). Arabinose had a final concentration of 0.1% and IPTG had a final concentration of 0.5mM. After the media change and addition of either arabinose and/or IPTG, cultures were incubated at 37C with shaking at 220rpm for an additional 3 hours, after which cells were pelleted by centrifugation (3500xg for 5min) for RNA harvest and qPCR analysis.



Collection of RNA

Cells were pelleted by centrifuging cultures at 3500xg for 5min, and supernatant discarded

RNA was isolated using RNA Miniprep Isolated Kit (Qiagen) following manufacturer’s instructions


cDNA First Strand Synthesis

1-8uL of RNA (3ug RNA)

1uL Random hexamer primers (ThermoFisher)

1uL 10mM dNTPs (ThermoFisher)

Volume adjusted to 10uL with DEPC-treated water

RNA/NTP solution was incubated at 65C for 5 in a BioRad MyCycler thermocycler, then the following were added:

2uL 10XRT buffer (ThermoFisher)

4uL 25mM MgCl2 (ThermoFisher)

2uL 0.1M DTT (ThemroFisher)

1uL RNaseOUT (ThermoFisher)

1uL SuperScript (ThermoFisher)

Reactions (20uL total volume) were run in BioRad MyCycler thermocycler at:

25C for 10min

50C for 50min

85C for 5min



All samples were run in triplicate

Individual samples were prepared as follows:

0.3uL cDNA

0.5uL forward primer

0.5uL reverse primer

10uL 2XSYBR Green Dye

8.7uL Water

Reactions were placed in a 96well qPCR plate (Applied Biosystems) and sealed with microplate adhesive film (Applied Biosystems)

Reactions were run on an Applied Biosystem 7500 Real Time PCR system

Data was analyzed using Excel (Microsoft)