Difference between revisions of "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 oC 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 ration

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

1. After electrophoresis, cut band of DNA-containing agarose and place in a sterile dialysis bag, clamped on one end.
2. 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)

3. 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
4. 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.
5. 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.
6. 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