Team:NYU-AD/Experiments

Exterminator Coli

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Transformation Protocol

1. Obtain tubes containing 50 uL of competent cells
2. Thaw tubes on ice for approximately 10 minutes
3. Add 1-5 uL of plasmid (containing 1 pg to 100 ng of DNA) to each tube
4. Carefully flick tube 4-5 times to mix, do not vortex. Keep tubes on ice for 15-30 minutes
5. Heat shock at 42 degrees c for 45 s
6. Place tubes on ice for 5 minutes, do not mix.
7. Add 950 uL of SOC media to each tube
8. Incubate tubes at 37 degrees C for 1 hour, shaking vigorously (250 rpm).
9. Mix well by inverting a few times
10. Pipette 50-100uL of outgrowth culture onto agar plates with the appropriate antibiotic(s)
11. Spread aliquot evenly across surface and incubate overnight at 37 degrees C.

Digestion And Ligation

Digestion

1. Add 38ul of water
2. Add 5ul of your plasmids
3. Add 5ul of NEB Buffer 2.1
4. Add 1 ul each of restriction enzymes
5. Incubate at 37˚C for 15mins
6. Inactivate the enzyme by incubating at 80˚C for 20mins

Ligation

1. Add 5ul each of digest to 2ul of 10x Ligase reaction buffer
2. Add 1ul of T4 DNA ligase
3. Add 7ul of water
4. Incubate the mix for 30mins at 16˚C
5. Inactivate by incubating at 65˚C for 10mins

Following ligation, proceed to the transformation of the ec.oli competent cells with each of ligated products. Follow the transformation protocols you previously were given and plate your transformations on the appropriate selective media. Use ccdb resistant cells when need be.

QIAprep Spin Miniprep Kit:

1. Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000
2. Add provided RNAse A solution to Buffer P1, mix, store at 2-8˚C
3. Add ethanol ( 96-100%) to Buffer PE before use
4. Pellet bacterial overnight culture by centrifugation at >8000rpm (6800 x g) for 3 min at room temperature ( 15-25 ˚C)
5. Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube
6. Add 250ul of Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow lysis to continue for more than 5 min. If using LyseBlue reagent, the solution will turn blue
7. Add 350ul of Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution should turn colourless
8. Centrifuge for 10 min at 13,000 rpm ( 17,900 x g) in a table-top microcentrifuge
9. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60secs and discard the flow through.
10. Recommended: Wash the QIAprep spin column by adding 500ul Buffer PB. Centrifuge for 30-60secs and discard the flow through.
11. Wash the QIAprep spin column by adding Buffer PE. Centrifuge for 30-60seconds and discard the flow through. Transfer the QIAprep spin column to a collection tube
12. Centrifuge for 1 min to remove residual wash buffer
13. Place the QIAprep spin column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50ul Buffer EB ( 10mM Tris.Cl, pH 8.5) or water to the centre of the QIAprep spin column, let it stand for 1 min and centrifuge for 1 min.

Nanodrop Operating Protocol

1. Put 1-2 μL of deionized water onto the measurement pedestal; have the sampling arm open.
2. After putting down the sampling arm, start the Nanodrop software and select the following: Start → Programs → NanoDrop → ND-1000.
3. With the software running, click OK to the following message on the screen: “Ensure pedestals are clean. Click OK to initialize instrument”.
4. The message “Initializing Spectrometer – please wait” will appear. When this message is gone, the equipment is ready. The data will be automatically recorded into the archive file.
5. Wipe pedestals with a laboratory wipe.
6. Put on a reference blank onto the lower measurement pedestal.
7. With the sampling arm down again, make sure the sample column is between the upper and lower measurement pedestals to make the spectral measurement. Store the blank reference.
8. Confirming that the blank solution and the solvent of the sample are the same material, repeat this blanking process until the spectrum is within 0.005 A (1mm path).
9. Analyze the sample, making sure the solute is mixed well into the solvent.
10. Clean off all surfaces thoroughly to avoid cross-contamination.

https://www.sdstate.edu/aes/FGCF/upload/NanodropFGCFProtocol.pdf

Gel Electrophoresis

1. Dissolve the agar soltion and cool it, then pour the gel into the gel electrophoresis template, or use the pre-prepared gels.
2. Load samples in the wells of the gel.
3. Place the gel into the electrophoresis chamber with the wells closest to the negative (black) electrode.
4. Prepare the salt solution and add it to the chamber.
5. Secure the lid and connect the electrode leads to the battery.
6. Turn on the power supply and adjust the voltage (50 – 100 v)
7. Run the gel for 5 – 10 minutes, observing the movement of the samples moving through the agar.
8. Turn off power supply, disconnect electrode leads, and remove chamber lid.
9. Remove gel from electrophoresis chamber for examination.
10. Discard gel in the trash and clean the chamber and template with water; dry thoroughly.

(http://www.colorado.edu/Outreach/BSI/pdfs/electrophoresis_teacher.pdf)

Lactic Acid Assay

Lactate Standards for Colorimetric Detection

Dilute 10 μL of the 100 nmole/ μL Lactate standard with 990 μL of Lactate Assay Buffer to generate a 1 nmole/μL standard solution. Add 0, 2, 4, 6, 8 and 10 μL of the 1 nmole/μL Lactate standard into a 96 well plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Lactate Assay Buffer to each well to bring the volume to 50 μL.

Lactate Standards for Fluorometric Detection

Prepare a 1nmole/μL standard solution as for the colorimetric assay. Dilute 1 μL of the 1nmole/μL standard solution with 990 μL of the Lactate Assay Buffer to make a 0.01 nmole/μL standard solution. Add 0, 2, 4, 6, 8 and 10 μL of the prepared 0.01 nmole/μL standard solution into a 96 well plate, generating 0 (blank), 20, 40, 60, 80, and 100 pmole/well standards. Add Lactate Assay Buffer to each well to bring the volume to 50 μL.

Sample Preparation

Both the colorimetric and fluorometric assays require 50 μL of sample for each reaction (well).

Tissue or cells can be homogenized in 4 volumes of the Lactate Assay Buffer. Centrifuge the samples at 13, 000 x g for 10 minutes to remove insoluble material. Samples should be deproteinized with a 10 kDa MWCO spin filter to remove lactate dehydrogenase. The soluble fraction may be assayed directly.

Serum samples (0.5-10 μL/assay) can be assayed directly by adding in duplicate to 96 well plate. If lactate dehydrogenase activity is present, samples should be deproteinized with a 10kDa MWCO spin filter.

Bring samples to final volume of 50 μL/well with Lactate Assay Buffer.

For unknown samples, it is suggested to test several sample volumes to make sure the readings are within the standard curve range.

Note: Lactate dehydrogenase (LDH) will degrade lactate. There, samples containing LDH (such as culture medium or tissue lysate) should be kept -80 degrees Celsius for storage, and filtered through a 10kDa cut-off spin filter. Complete medium containing FBS should be deproteinized due to high LDH content.

Assay Reaction

1. Set up the Master Reaction Mix according to the scheme in Table 1. 50 μL of the Master Reaction Mix is required for each reaction (well).



Reagent	Master Reaction Mix
Lactate Assay Buffer	46μL
Lactate Enzyme Mix	2 μL
Lactate Probe	2 μL



2. Add 50μL of the Master Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 30 minutes at room temperature. Protect the plate from light during the incubation.
3. For colorometric assays, measure the absorbance at 570 nm (A570). For fluorometric assays, measure fluorescence intensity ( ex = 587 nm).