Team:NCKU Tainan/Protocol

Protocols

Genomic DNA Extraction

GeneMark Tissue&cell Genomic DNA Purification Kit

  1. Gram-negative bacteria: Centrifuge cells (< 2 x 109 ) at 14,000 xg for 2 min., carefully remove the supernatant and resuspend bacterial pellet in 200 μl Extraction Solution. Mix by vortexing or pipetting. Proceed to Step 2.

    Gram-positive bacteria: Centrifuge cells (< 2 x 109 ) at 14,000 xg for 2 min., and carefully remove the supernatant. Incubate the pellet in - 80℃ for 5 min. (or in liquid nitrogen for 30 seconds), and then immediately incubate the tube in 37℃ for 2 min. Resuspend the pellet in 180 μl of Lysozyme Solution. Mix by vortexing or pipetting. Incubate at 37 ℃ for 30 min. and add 200 μl Extraction Solution. Proceed to Step 2.
  2. Add 20 μl of Proteinase K Solution to the microcentrifuge tube, mix by vortexing.
    • Do not premix Extraction Buffer and proteinase K solution before use to prevent proteinase K from undergoing self-digestion without substrate.
  3. Implemented Bio-Safety to our devices.
  4. Incubate samples at 56℃ in water bath or incubator for 0.5~3 hours or longer until complete lysis of tissue (time varies depending on sample size), vortex 5~10 seconds at frequent intervals during incubation.
    Sample Time
    Cultured cells from tissue (< 5 x 106 ) 0.5 ~ 1 hours
    Animal or insect tissue (< 25 mg), for spleen (< 10 mg) 1 ~ 3 hours
    Bacteria (< 2 x 109) 0.5 ~ 1 hours
    Yeast cell (< 5 x 107 ) 0.5 ~ 1 hours
  5. To remove tissue debris, centrifuge at 14,000 xg for 5 min. after lysis is complete. Transfer the clear lysate to a new 1.5 ml microcentrifuge tube.
  6. Optional: If high quality DNA (no RNA contamination) is desired, add 4 μl RNaseA solution and incubate at RT for 5 min.
  7. Add 200 μl of Binding Solution to the lysate, mix by vortexing.
  8. Incubating at 70℃ water bath or heating block for 10 min.
    • A white precipitate may form after addition of Binding Solution, which dissolves during incubation at 70℃. For some tissues, the mixture may not turn clear (e.g. brain tissue), however, this does not affect DNA binding. The mixture will turn clear after addition of ethanol.
  9. Add 200 μl Ethanol, mix thoroughly by vortexing, and transfer the mix (including any precipitates) to spin column mounted into a collection tube. Centrifuge at top speed for 1 min.
    • A precipitate may form after addition of ethanol, please do not spin. Transfer the mix (including the solution and precipitate) to the column directly.
  10. Discard the filtrate, add 300 µl of Binding Solution to the column and centrifuge at top speed for 1 min.
    • This step is essential for removal of endonuclease to prevent Contamination.
  11. Discard the filtrate, add 700 µl of Wash Solution and centrifuge at top speed for 1 min. Repeat this step once more.
  12. Discard the filtrate and centrifuge for additional 5 min. at top speed to remove residual trace of ethanol.
    • If centrifugation speed is lower than 12,000 xg or residual ethanol must be removed completely, incubate the spin column in a heat oven (60~65℃) for 5 min. to evaporate all of the ethanol.
  13. Transfer the spin column into a new microcentrifuge tube and add 100 ~ 200 μl preheated (60~65℃) Elution Solution or H2O (pH 7.0 ~ 8.5) into the column and wait for 1~2 min.
  14. Centrifuge at top speed for 1 min. to elute the DNA. Store the eluted DNA at -20℃.
    • Repeating elution once will increase DNA yield by 10 ~ 15 %, though DNA will be diluted.

Plasmid Extraction

FAVORGEN FavorPrepTM Plasmid DNA Extraction Mini Kit

  1. Transfer 2 ml of well-grown bacterial culture to a centrifuge tube.
  2. Centrifuge the tube at 18,000 xg for 3 minute to pellet the cells and discard the supernatant completely.
  3. Add 200 µl of FAPD1 Buffer (RNaseA added) to the cell pellet and resuspend the cells completely by pipetting.
    • Make sure that RNaseA has been added into FAPD1 Buffer when first use.
    • No cell pellet should be visible after resuspension of the cells.
  4. Add 200 µl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times. Incubate the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells.
    • Do not vortex, vortex may shear genomic DNA. If necessary, continue inverting the tube until the lysate become clear.
    • Do not proceed with the incubation over 5 minutes.
  5. Add 300 µl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to neutralize the lysate.
    • Invert immediately after adding FAPD3 Buffer will avoid asymmetric precipitation.
  6. Centrifuge at 18,000 xg for 10 min. to clarify the lysate. During centrifugation, place a FAPD Column in a Collection Tube.
  7. Transfer the supernatant carefully to the FAPD Column and centrifuge at 18,000 xg for 1 minute. Discard the flow-through and place the column back to the Collection Tube.
    • Do not transfer any white pellet into the column.
  8. Add 400 µl of W1 Buffer to the FAPD Column and centrifuge at 18000 xg for 1 minute. Discard the flow-through and place the column back to the Collection Tube.
  9. Add 600 µl of Wash Buffer to the FAPD Column and centrifuge at 18,000 xg for 1 minute. Discard the flow-through and place the column back to the Collection Tube.
    • Make sure that ethanol (96 ~ 100 %) has been added into Wash Buffer when first use.
  10. Centrifuge at 18,000 xg for an additional 5 minutes to dry the FAPD Column.
    • Important step! The residual liquid should be removed thoroughly on this step.
  11. Place the FAPD Column to a new 1.5 ml microcentrifuge tube.
  12. Add 65 µl of Elution Buffer or ddH2 O to the membrane center of the FAPD Column. Stand the column for 3 minute.
    • Important step! For effective elution, make sure that the elution solution is dispensed on the membrane center and is absorbed completely.
    • Do not elute the DNA using less than suggested volume (50 µl). It will lower the final yield.
  13. Centrifuge at 18,000 xg for 3 minute to elute plasmid DNA and store the DNA at -20 ℃.

PCR Clean-Up & Gel Extraction

Sample Preparation

  1. PCR Cleanup
    1. Added 500 μl of the Buffer B to 100 μl of the PCR product and mix by vortex.
  2. Gel Extraction
    1. Excised the DNA fragment from the agarose gel.
    2. Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube.
    3. Added 500 μl of the Buffer B to the sample and mixed by vortex. Incubate at 60℃ for 10 minutes (or until the gel slice has completely dissolved).
    4. During the incubation, mixed by vortexing the tube every 2~3 minutes.
    5. Cooled the dissolved sample mixture to the room temperature.

    3. Binding

  3. Placed a PG Column in a Collection Tube. Apply the supernatant to the PG Column by decanting or pipetting.
  4. Centrifuged at 18,000 xg for 30 seconds.
  5. Discarded the flow-through and place the PG Column back into the same collection tube.

    6. Wash

  6. Added 400 μl of the Buffer W1 into the PG Column.
  7. Centrifuged at 18,000 xg for 30 seconds.
  8. Discarded the flow-through and place the PG Column back into the same collection tube.
  9. Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.
  10. Centrifuged at 18,000 xg for 30 seconds.
  11. Discarded the flow-through and place the PG Column back into the same collection tube.
  12. Centrifuged at 18,000 xg again for 2 minutes to remove the residual Buffer W2.

    13. Elution

  13. To elute the DNA, placed the PG Column in a clean 1.5 ml microcentrifuge tube.
  14. Added 50~200 μl of the Buffer E or H2O (pH is between 7.0 and 8.5) to the center of each PG Column, let it stand for 2 minutes, and centrifuge at 18,000 xg for 2 min.
    15. NOTE: Check the buffers before the use for salt precipitation. Redissolve any precipitate by warming to 37℃.

PCR

  1. Gently mix the following reaction by pipetting and centrifuge briefly.
    20 μl system 50 μl system
    Template 12～20 ng 30～50 ng
    Forward primer 1.0 μl 2.5 μl
    Reverse primer 1.0 μl 2.5 μl
    dNTP 1.6 μl 4.0 μl
    10x Buffer 2.0 μl 5.0 μl
    DNA polymerase 0.2 μl 0.5 μl
    ddH2O Up to 20 μl Up to 50 μl

    Program of Taq(Ex Taq) DNA polymerase

    Temperature Time
    94 ℃ 3 min.
    94 ℃ (Denaturation) 40 sec 25～30 cycles
    55 ℃ (Annealing) 30 sec
    72 ℃ (Extension) Depend on sequence size (2 kbp/min. for Taq)
    72 ℃ 5 min.
    4 ℃ ∞
  2. Confirm the size of the digested product by gel electrophoresis.
  3. Gel purification of the target size.

Plasmid Construction

  1. Digestion(vector)
    Plasmid 200 ng 1000 ng
    EcoRI / SpeI 0.2 μl 1 μl
    XbaI / PstI 0.2 μl 1 μl
    10x Q. cut buffer 2 μl 5 μl
    ddH2O Up to 20 μl Up to 50 μl
    Digestion at 37℃ for 1hr.
  2. Digestion(insert)
    Plasmid 200 ng 1000 ng
    EcoRI / XbaI 0.2 μl 1 μl
    SpeI /PstI 0.2 μl 1 μl
    10x Q. cut buffer 2 μl 5 μl
    ddH2O Up to 20 μl Up to 50 μl
    Digestion at 37℃ for 1hr.
  3. Confirm the size of the digested product by gel electrophoresis.
  4. Gel purification of the target size.
  5. Ligation
    Vector (2 kbp) molar ratio = 1:3
    (can up to 1:10 depending on the DNA sizes)
    Insert (1.5 kbp)
    10x T4 DNA ligase buffer 1 μl
    T4 DNA ligase 0.5 μl
    ddH2O Up to 10 μl
  6. Transform the product by heat shock.

Competent Cell Preparation

  • For E. coli DH5α and MG1655 competent cell
  1. Streak out wild type E. coli on a plate (LB plate without antibiotics) overnight and pick one colony into 3 ml of media (LB) and grow overnight.
  2. Transfer 0.2 ml of starter culture into 10 ml of fresh LB with 10 mM MgCl 2 the next day and grow culture at 37 ℃.
  3. When the OD600 nm up to 0.35, put the cells on ice immediately.
  4. Spin the cells at 4℃for 10 minutes at 3000 rpm.
  5. Suspend the pellet on ice carefully with 4 ml chilly TB buffer
  6. Leave nicely suspended bugs on ice for 10 minutes.
  7. Spin the cells at 4℃ for 10 min. at 3000 rpm.
  8. Suspend the pellet on ice with 0.8 ml of TB buffer.
  9. Add 0.06 ml DMSO, and mix it
  10. Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with liquid nitrogen.
  11. Store the frozen cells in the -70℃ freezer.

Transformation Buffer (TB)

  • 10 mM PIPES
  • 15 mM CaCl2•2H2O
  • 250 mM KCl
  • Adjust pH to 6.7 by KOH.
  • Then add MnCl2•4H2O to final concentration 55 mM.
  • Filter the buffer and store at 4℃.
NOTE: Do not autoclave.

Griess Reagent Assay

Solution preparation

  1. Prepare the following solution for Griess reagent.
    1. N-(1- naphthyl)ethylenediamine dihydrochloride (Component A), 25 mL of a 0.1% (1 mg/mL) solution.
    2. Sulfanilic acid (Component B), 25 mL of a 1% (10 mg/mL) solution in 5% phosphoric acid.
    3. 0, 10, 20 and 50 μM Sodium nitrite/Potassium nitrate solution for standard curve.
    • All component should be stored at 2~6℃ and protected from light.
    • Mix together equal volumes of N-(1- naphthyl)ethylenediamine (Component A) and sulfanilic acid (Component B) to form the Griess Reagent.
    • Prepare sufficient reagent for immediate experiments only (100 µL per spectrophotometer cuvette, 20 µL per microplate well). Do not store the mixture for more than 8 hours.

    2. Standard curve

  2. Add 100 μl nitrite/nitrate standard solution, and 100 μl Griess reagent into 96-well plate, and gently pipetting.
  3. Scan the OD545 nm by SpectraMax 340PC384 Microplate Reader
  4. Plot the relation between OD545 nm and nitrite concentration as the standard curve.

    5. Sample test

  5. Cultivate theE. coliharboring BBa_K2275007 plasmid for 8 hr.
  6. Add nitrite/nitrate solution with different concentration into broth and centrifuge at 3000 rpm for 5 min. Take out 100 μl supernatant for blank test and then resuspend the residue broth.
  7. The residue broth continue incubating at 37℃ for 4 hr. As above, centrifuge at 3000 rpm for 5 min., then take out 100 μl supernatant as reacted sample.
  8. Add 100 μl Griess reagent into both blank and reacted sample. React preventing from light in RT for 5 min.
  9. Scan the OD545 nmby SpectraMax 340PC384 Microplate Reader
  10. Compare with the standard curve to calculate the concentration of nitrite/nitrate in supernatant.

Glutamate Colorimetric Assay Kit

Reagent reconstitution

  1. Glutamate Developer + 820 µl ddH2O
  2. Glutamate Enzyme Mix + 220 µl Assay Buffer
    • Store kit at -20℃, protected from light.
    • Allow buffer to warm to RT before use. Enzyme Mix should keep on ice while in use.

    Standard curve preparation

  3. 10 µl 0.1 M Glu standard + 990 µl Assay Buffer to 1 mM.
  4. Add diluted Glu standard solution to generate 0, 2, 4, 6, 8 and 10 nmole/well Gln in 96-well plate. Then adjust the volume to 50 µl/well with Assay Buffer.

    5. Reaction Mix

  5. Mix enough reagents for the number of assays to be performed. For each well, prepare a total 100 µl Mix containing
    Reaction Mix Background Control Mix
    90 µl Assay Buffer 92 µl Assay Buffer
    8 µl Glutamate Developer 8 µl Glutamate Developer
    2 µl Glutamate Enzyme Mix －
  6. Add 100 µl of the Reaction Mix to each well containing the Glutamate Standard and test samples. To the background control well, add 100 µl of background control mix. Mix well.

    7. Measurement

  7. Incubate the reaction for 30 min. at 37℃, protected from light. Measure OD450 nmby SpectraMax 340PC384 Microplate Reader.

Glutamine Colorimetric Assay Kit

Reagent reconstitution

  1. Hydrolysis Enzyme Mix + 220 µl Hydrolysis Buffer
  2. Development Enzyme Mix + 220 µl Development Buffer
  3. Developer + 220 µl Developer Buffer
  4. Gln Standard + 100 µl ddH2O to 10 mM
    • Store kit at -20℃, protected from light.
    • Allow buffer to warm to RT before use. Enzyme Mix should keep on ice while in use.

    Standard curve preparation

  5. 10 µl 10mM Gln standard + 90 µl ddH2O to 0.1 mM.
  6. Add diluted Gln standard solution to generate 0, 2, 4, 6, 8 and 10 nmole/well Gln in 96-well plate. Then adjust the volume to 40 µl/well with ddH2O.

    7. Hydrolysis Mix

  7. Add 2 µl Hydrolysis Enzyme mix to the Standard and Sample wells as follows
    Standard/Sample Background
    Hydrolysis Enzyme Mix 2 µl －
    Hydrolysis Buffer 8 µl 10 µl
  8. Mix well. Adjust the volume to 50 µl/well with ddH2O if necessary. Incubate for 30 min. at 37℃.
    • For samples having high glutamate levels, add 10 µl of Hydrolysis Buffer to background control well(s). Adjust the volume to 50 µl/well with ddH2O & incubate for 30 min. at 37℃.

    Reaction Mix

  9. Mix enough reagents for the number of assays to be performed. For each well, prepare 50 µl Reaction Mix containing
    Reaction Mix
    Development Buffer 46 µl
    Development Enzyme Mix 2 µl
    Developer 2 µl
  10. Mix well. Add 50 µl of the Reaction Mix to each well containing Standards, samples and Background Control(s). Mix well.

    11. Measurement

  11. Incubate at 37℃ for 60 min., protected from light. Measure OD450 nm by SpectraMax 340PC384 Microplate Reader.

Reference

GeneMark Tissue&cell Genomic DNA Purification Kit
FAVORGEN FavorPrepTM Plasmid DNA Extraction Mini Kit
GeneDirex PCR Clean-Up & Gel Extraction Kit
Griess reagent assay
BioVision Glutamate Colorimetric Assay Kit
BioVision Glutamine Colorimetric Assay Kit