Team:Stony Brook/Experiments
Protocols and Experiments
Luria Bertani Broth Protocol
This protocol makes 1L of LB and can be adjusted as needed.
Combine and mix using a magnetic stir bar on low heat. Autoclave for 20 minutes on a liquid cycle.
Luria Bertani Agar Plate Protocol
Combine and mix using a magnetic stir bar on low heat. Autoclave for 20 minutes on a liquid cycle. Allow LB agar to cool to about 55°C before adding antibiotic. Pour into plates.
Nanodrop Protocol
• Select Nucleic Acid
• Clean pedestal, drop 1ul water, press okay
• Blank with 1ul of room temperature water
• Measure with 1ul of DNA
• 260/280 ratio should be near 2
Transformation Protocol
• Thaw competent E. coli cells on ice and pipette 50 ul of cells into a Eppendorf tube
• Add 1-5ul containing 1 pg-100 ng of plasmid DNA and flick tube 4-5 times
• Blank with 1ul of room temperature water
• Place tube on ice for 30 minutes
• Heat shock at 42°C for 30 seconds
• Place tube on ice for 5 minutes
• Add 950 ul room temperature SOC to mixture
• Incubate at 37°C for 60 minutes at 250 rpm
• Spread 50-100ul onto a plate and incubate overnight at 37°C
Restriction Enzyme Digest Protocol
• Make negative controls without restriction enzymes
• Incubate at 37°C for at least one hour
Ligation Protocol
• Use a 1:3 vector to insert ratio
• Add T4 DNA Ligase last
• Pipette the mixture up and down microfuge
• Incubate overnight at 16°C
PCR Purification Protocol
We used the PCR and Gel Clean-up kit by Enzo Life Sciences. Their PCR Purification protocol can be found on page 7 of their product manual.
Miniprep Protocol
We used Qiagen’s QIAprep® Spin Miniprep Kit. Their protocol can be found here.
Gel Extraction Protocol
We used NEB’s Monarch DNA Gel Extraction Kit. Their protocol can be found here.
Gel Electroelution Protocol
Thank you to Dr. Raafat El-Maghrabi for sharing his protocol with us
• After electrophoresis, cut band of DNA-containing agarose and place in a sterile dialysis bag, clamped on one end.
• Add 0.4 ml of TE buffer to bag, remove as much air from the bag as possible, move agarose band to one side of the bag, and close with a second clamp (see figure)
• Place dialysis bag in electrophoresis cell so that the DNA in the agarose is closest to, and facing the elution buffer and the anode. The dialysis bag should be just submerged in electrophoresis buffer
• Electrophorese at ~100v, occasionally monitoring the migration of the DNA under UV-light, until DNA has migrated out of agarose and lines up at the opposite edge of the bag. Switch polarity of the electrodes and reverse electrophoresis for a minute to elute any dialysis bag-bound DNA.
• Transfer the 0.4 ml of buffer containing the DNA to an Eppendorf tube, and rinse bag with 0.1 ml of additional buffer. Extract once with 0.5 ml phenol and then with 0.5ml CHCl3, and precipitate by adding 50μl of 3M sodium acetate and 1.2 ml of 95% ethanol.
• After ~30min at -70°C, microfuge for 15min, rinse pellet with 0.5ml 70% ethanol and dry in Speed-Vac. Dissolve in 10-20μl of TE0.1 and quantitate by electrophoresing 1-2μl in agarose next to a known STD (1 μg of λ /StyI gives, in ascending size order; bands of 10, 20, 30, 40, 55, 70, 90, 130, 160 and 400 ng of ds DNA).
Agarose Gel Preparation
• Add 0.6 grams of 1% agarose to an Erlenmeyer flask (0.9 g for a 1.8% gel).
• Fill to 50 mL with 1x TAE
• Microwave for 30 second intervals until bubbling, cool slightly
• Add 2uL of ethidium bromide and mix
• Pour into gel box, avoid air bubbles.
• Add comb
• Allow to cool
Lysis Buffer Protocol
• 50 mmol Sodium Phosphate
• 300 mmol Sodium Chloride
• 10 mmol Imidazole
• Fill to 1L with Milli-Q water
• Adjust the pH to 7.5 using HCl
Wash Buffer Protocol
• 50 mmol Sodium Phosphate
• 300 mmol Sodium Chloride
• 25 mmol Imidazole
• Fill to 1L with Milli-Q water
• Adjust the pH to 7.5 using HCl
Elution Buffer Protocol
• 50 mmol Sodium Phosphate
• 300 mmol Sodium Chloride
• 250 mmol Imidazole
• Fill to 1L with Milli-Q water
• Adjust the pH to 7.5 using HCl
Protein Purification Protocol
1. Add 20-30 mL of Lysis Buffer and allow BL21 E. coli pellet to thaw
1.1 Vortex as needed and eliminate all clumps
2. Cell Lysis (Freeze-thaw or Sonication)
2.1 Add 1mg/mL lysozyme and leave on ice for 30 minutes
2.2 Place in -80oC freezer until frozen
2.3 Thaw completely in water bath
2.4 Repeat cycle 2 more times, then add 5uL of DNAse
2.5 Incubate for 30 minutes on ice
3. Centrifuge for 45 minutes at 15,000 rpm
3.1 Collect the supernatant in a fresh tube and save a sample of the pellet.
4. Column
4.1 Equilibrate the Ni-NTA resin
4.1.1 Add the Ni-NTA resin to the column
4.1.2 Add 20-30 mL of lysis buffer to the column
4.1.3 Discard flow through
4.2 Add the lysate to the column and collect the flow through in a 50 mL falcon tube.
4.2.1 Add the flow through to the column once more.
4.3 Wash #1
4.3.1 Add 20 mL of lysis buffer to the column and collect the flow through in a fresh 50 mL falcon tube
4.4 Wash #2
4.4.1 Add 20 mL of wash buffer to the column and collect the flow through in a fresh 50 mL falcon tube
4.5 Elution
4.5.1 Add 10 mL of elution buffer. Collect flow through in 0.5mL fractions
4.5.2 Add Bradford reagent to a 20uL elution fraction sample to check for protein
5. Analysis
5.1 Check protein concentrations of the elution fractions using the Nanodrop A280 setting
5.2 Run an SDS-PAGE to check for protein in elution fractions, washes, supernatant, and cell pellet.
Preparing and Running an SDS-PAGE
1. Prepare a sample of the cell pellet, cleared lysate, washes, and 10 elution fractions.
a.Cell Pellet 1-2 uL, 18 uL of Milli-Q water, 7 uL 4x SDS-PAGE loading buffer
b.Cleared lysate 1-2 uL, 18 uL of Milli-Q water, 7 uL 4x SDS-PAGE loading buffer
c.Washes (1 & 2) 5-10 uL, 10-15 uL of Milli-Q water, 7 uL 4x SDS-PAGE loading buffer
d.Elution Fractions (1-10) 20 uL, 7 uL 4x SDS-PAGE loading buffer
e.Add 3 uL of BME to each sample
2. Boil prepared samples for 3 minutes
3. Load samples and run gel for 45 minutes to 1 hour at 100 mA in 1x running buffer.
4. Place gel in a tupperware container and cover it with coomassie blue stain. Allow it to stain for 1-2 hours on a rocker.
5. Pour out Coomassie stain and rinse with Milli-Q water. Add destaining solution (methanol, glacial acetic acid, and water) and allow it to rock for 1-2 hours.
6. View the gel.
Dot Blot Procedure
1. Incubate the membrane in methanol for 5 minutes before beginning
2. Set up the membrane with circles for each of the samples
3. Place a 5 microliters of each sample in each circle (add positive, ADE, and negative, untransformed BL21 cells, control as well)
4. Let the membrane sit for a half hour to absorb the samples
5.Block the membrane for an hour with a 2% powdered milk-TBS buffer for an hour at room temperature
6.Wash the membrane with TBS buffer to clean the milk solution off and then add a 1:2000 dilution of Anti-his antibody in TBS buffer and rock at 4°C overnight
7.Add the horseradish peroxidase to the membrane and let sit at room temperature for an hour
8.Wash the membrane with TBS buffer before reading
MRSA Testing:
Our MRSA and S. aureus testing took place during a 3 day period in order to limit exposure to the bacteria in the laboratory and minimize risk.
Rehydration of freeze-dried bacteria from ATCC under laminar flow hood:
• Heat the tip of the glass outer vial using a Bunsen burner
• Crack the glass of the outer vial by squirting water on it
• Strike with a file to remove the outer vial glass
• Raise cotton plug with forceps and rehydrate the bacteria
• Dispose of all broken glass and packing materials in a designated sharps container
Tuesday August 24th
• Biosafety Level Two Restriction was placed on the laboratory space
• Small liquid cultures of Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus (MRSA) were made so that enough bacteria was present for the Minimum Inhibition Concentration (MIC) Assay and the Spot on Lawn Assay
• 2 Mueller Hinton Plates were then coated with the Staph. aureus and 2 Mueller Hinton plates were coated with MRSA to prepare for the Spot on Lawn Assay
Wednesday August 25th
• 13 small liquid cultures were prepared of Staph aureus and 13 more were created for MRSA.
• Each liquid culture had 50 μL of a bacteriocin or fusion bacteriocin added to the tube according to the following chart. 13 was left as a negative control in both groups
• For the Spot on Lawn assay, the plates were divided into 6 parts and each part had a small sterile paper circle with the bacteriocin or fusion bacteriocin placed in it. The placement of bacteriocins was similar to that of the MIC assay (1-12)
Thursday August 26th
• The plates and the cultures were analyzed and no change was observed in terms of growth for the MIC assay and no zone of inhibition was observed for the spot on lawn assay.
• All MRSA and Staph. aureus was transferred to a flask, covered, placed in an autoclave bag, and killed through autoclaving.
• The laboratory was disinfected using several cleaning agents and antimicrobial agents to ensure that the area was free of any possible dangerous microbes. The hood, after being thoroughly disinfected, was left with the UV light on for 30 minutes to kill anything remaining in it.
• All disposable materials used in the assays were disposed of properly (Masks, gowns, tips, etc.)
• The lab was stripped of its BSL 2 status and returned to its regular BSL1 status.
