Cyanine Dye Purification

主导提醒:Preparation of salt buffers The buffers are used for both HPLC
purification and Sep-Pak purification.High salt Preparation of salt
buffers

基本提醒:CyanineDyePurificationProtocolPreparation of salt buffersThe
buffers are used for both HPLC p

主导提醒:Reagents & EquipmentsOligo-dT – Roche dNTPs KugaNasin – Promega
SuperscripReagents & Equipments

High salt buffer :

CyanineDyePurificationProtocol

  • Oligo-dT – Roche
  • dNTPs
  • RNasin – Promega
  • Superscript II – Life Technologies
  • Qiaquick PCR purification system – QIAGEN
  • 22 x 50 mm cover slips
  • Cot-1 DNA – Life Technologies
  • Poly dA – Roche
  • Cy3 and Cy5-dUTP
  • 0.5M EDTA
  • 2M NaOH
  • 1M HCl
  • 1M Tris pH 8
  • 100mM sodium acetate
  • 20x SSC
  • 20% SDS
  • Deionized Formamide
  • MilliQ Water
  • Array chip
  • Hybridisation chamber
  • Dnase-, RNase-free 0.5 and 1.5 ml eppendorf tubes
  • Plastic slide holders
  • 2 litre beakers
  • 1M triethylammonium acetate, pH 8.0
  • 10% acetonitrile

Preparation of salt buffers

High salt buffer :

  • 1M triethylammonium acetate, pH 8.0
  • 10% acetonitrile

Low salt buffer :

  • 25mM triethylammonium acetate, pH 8.0
  • 10% acetonitrile

HSB, 800 mL

  • To 500mL ddH2O, add approx. 25-30 mL glacial acetic acid
  • Slowly add 111.3 mL HPLC grade triethylamine; the reaction is
    exothermic; the solution will become cloudy – wait until it clears
    before adding more)
  • Add dropwise an additional 10-15 mL acetic acid to adjust the pH to
    8.0.
  • Add 80 mL acetonitrile
  • Adjust the final volume to 800 mL with ddH2O.

LSB, 1000 mL

  • To 25 mL HSB, add 100 mL HPLC grade acetonitrile
  • Raise final volume to 1L with ddH2O

*Filter, and then sparge both buffers before use. Makes enough
for approx. 9 runs & 2 purges.


Low salt buffer :

Desalting the sample for Sep-Pack Purification

Materials:

  • HPLC grade acetonitrile
  • 20 ml ddH2O, 0.22 mm filtered
  • 15 mM TEAA
  • Sep-Pak cartridges, C18 ; 1 column per cy dye. Can be reused twice.
  • 12 mL syringe

Pre-load protocol:

  1. Wash Sep-Pak with 10 mL of acetonitrile.
  2. Wash with 5 mL of water.
  3. Wash with 10 mL of 15 mM TEAA.

Sample Preparation, Loading, and Elution:

  1. The column may be loaded up to 150 A260 units of sample ,
    lyophilized down to 3-5 mL .
  2. Add 1 M TEAA to the sample, to a concentration of 15 mM.
  3. Load the sample onto the column. The dye should be visible near the
    top of the column. Wash the column with 5 mL of 15 mM TEAA.
  4. Elute with 3 mL of acetonitrile. Discard the initial colorless
    drops, collect the colored eluent into a 2 mL centrifuge tube, and
    discard the remaining solution. The collected volume will be
    approximately 1.0-2.0 mL. Some colored material will rema in on the
    column after elution.

Immediately wash and process any subsequent lots, reusing the column
only twice for each lot. If the column is allowed to sit for more than 5
minutes, yields may decrease 5-10% or more. Avoid reusing the column
more than two times , as yields may begin to diminish. Pool the lots
together, check UV absorbance, and calculate yield. Expected yields for
this stage are 70-80% for either Cy dye.

HPLC Preparation

  1. Lyophilize the entire Sep-Pak purified material, keeping the dyes
    separate . Resuspend with a small volume of 0.2 M TEAA.
  2. Centrifuge the sample through a 0.45 mm filter.
  3. Check the absorbance at 260 nm and, if necessary, dilute the sample
    down to a concentration that will not exceed the capacity of the
    column column listed bel ow). Typically a 1 ml injection loop is
    used to inject a 10 mM sample of unlabeled dNTPs + Cy-dUTP.

LABELLING

  • 25mM triethylammonium acetate, pH 8.0
  • 10% acetonitrile

HPLC Purification

Column used: Dionex NucleoPac™ 9 ´ 250 PA-100; P/N 43011 HPLC Equipment:
Perkins Elmer Series 200 LC Pump, Perkins Elmer UV/Vis Detector LC 295

  1. Equilibrate column in buffer system, 3 mL/min flow rate

1.1. 5 min low salt buffer or A1.2. 2 min gradient to 100% high salt
buffer or B1.3. 5 min HSB or B1.4. 2 min gradient to 60% LSB or A1.5. 5
min 60% LSB or A

  1. Load sample into injection loop. See “Final Considerations” on the
    next page for sample lot size and other issues.

  2. Run gradient

3.1. 5 min 60% LSB, 40% HSB3.2. 10 min gradient to 80% HSB3.3. 1 min
gradient to 100% HSB3.4. 10 min 100% HSB3.5. 1 min gradient to 60%
LSB3.6. 5 min 60% LSB


  1. Mix the following:

    17 ul 25 ug total RNA or 1-5 ug of mRNA
    1 ul RNasin
    2 ul Oligo dT

    Note: if the total volume of the RNA is > 17 ul add 1 ul of
    RNasin and speedivac down to 18 ul without heat.

  2. Heat to 70oC for 10 min. Cool on ice for 1 min.

  3. Prepare the following:

    8 ul 5x RT buffer
    4 ul DTT
    1 ul dATP, dCTP, dGTP
    2 ul dTTP
    2 ul Cy3 or Cy3-dUTP – eg Cy3 for RNA from non-stimulated cells and Cy5 for RNA from stimulated cells
  4. Add the above mix to the RNA + oligo dT mix. Pulse spin and return
    to the ice. Add 1.5 ul of Superscript II. Incubated for 60 min at
    42oC.

  5. Add another 1 ul of Superscript II and incubate for a further 30
    min. Then quench the reaction on ice for 1 min.
  6. Add 1 ul of 0.5M EDTA and 2 ul of 2M NaOH. Heat to 65oC for 10 min.
    Hydrolysis destroys RNA.
  7. Add 4 ul 1M HCl and 4 ul 1M Tris pH 8.
  8. Add 17 ul of 100mM sodium acetate to each reaction.

HSB, 800 mL

Final Preparative Procedures

For a preparative run of 150 nmoles Cy-dUTP , the recovered fractions of
purified Cy-dUTP is eluted in 5 ml of volatile TEAA buffer that can be
removed by lyophilization.

Lyophilization and Redilution

  1. Pool fractions of each Cy dye into a 50 mL conical tube. Punch holes
    into the cap using an 18-gauge needle. Avoid exceeding more than
    half the volume of the tube, as the samples may have a tendency to
    splatter during degassing or travel up the tube during
    lyophilization.
  2. Freeze the samples on dry ice and transfer them to a lyophilization
    chamber. Wrap the chamber with foil to protect the samples from
    light.
  3. Lyophilize the samples to completion . HPLC-purified samples will
    have the appearance of a residue until most of the TEAA is removed.

    Note: Steps 4 & 5 are for HPLC-purified samples only.

  4. Dilute the samples back up to 8-10 mL with 0.22 mm filtered ddH2O
    and lyophilize until the samples again resemble residue. This time,
    it may require up to 24 hours of lyophilization.

  5. Repeat Step 4. Lyophilization is complete when the sample has the
    appearance of a film and/or specks of solid residue. Resuspend the
    sample in a small volume of 10 mM phosphate buffer, pH 7.0. Bear in
    mind that some of the Cy-dUTP will coat the upp er regions of the
    tube walls and may not be readily visible. Check absorbance of the
    sample. A560/660/A260 ratios should be around 17 for cy 3 and 25 for
    cy 5.

Expected overall yields: cy 3: 50-70%; cy 5: 45-65%.


PURIFICATION

  • To 500mL ddH2O, add approx. 25-30 mL glacial acetic acid
  • Slowly add 111.3 mL HPLC grade triethylamine; the reaction is
    exothermic; the solution will become cloudy – wait until it clears
    before adding more)
  • Add dropwise an additional 10-15 mL acetic acid to adjust the pH to
    8.0.
  • Add 80 mL acetonitrile
  • Adjust the final volume to 800 mL with ddH2O.

Final Considerations

Currently 150nmol of Cy-dUTP contained in a 10 mM mixture of labeled and
unlabeled dNTPs is loaded onto the column listed above, depending on the
contents and concentration of the filtrate; PCR flow-through samples
tend to be cleaner and may allow for larger injection lots. Thus, the
flow-through from PCR and RT reactions may be kept separate so that they
can be processed separately. The Sep-Pak purification desalts the sample
and is likely to remove other impurities present in the sample. HPLC
purif ication can still be performed without Sep-Pak purification, but
salt content prohibits injection of more than 50nmol. Furthermore,
yields tend to be lower if the sample is not desalted prior to HPLC
injection.

Amended protocol for Qiaquick PCR purification kit

LSB, 1000 mL

  1. Pool Cy3 and Cy5 reactions and add 425 ul PB buffer. Load the
    mixture onto column.
  2. Spin at 14K for 1 min. Discard eluate.
  3. Wash column with 650 ul of PE buffer.
  4. Spin at 14K for 1 min. Discard eluate.
  5. Spin at 14K for 1 min.
  6. Transfer column to fresh collection tube
  7. Add 40 ul of EB elution buffer to the column. Let stand 1-2 min.
    Elute purified cDNA by spinning at 14K for 1 min.
  • mgm5808美高梅,To 25 mL HSB, add 100 mL HPLC grade acetonitrile
  • Raise final volume to 1L with ddH2O

HYBRIDISATION

*Filter, and then sparge both buffers before use. Makes enough for
approx. 9 runs & 2 purges.

  1. To a fresh 0.5 ml tube add:

    10 ul Cot-1 DNA
    2 ul poly dA
    40 ul purified cDNA probe

    Speedivac down to a final volume of 11 ul.

  2. Add the following in order

    8 ul 20x SSC
    20 ul Deionized formamide
    1 ul 20% SDS

    Mix, spin down, and heat sample to 95oC for 5 min

  3. Incubate the labelled target at 45oC for 0.5-1.5 hr prior to placing
    on array. Temperature of probe is critical. Too hot leads to a
    scanning artefact, too cold and probe will precipitate. Make sure
    hybridisation chamber, slides and coverslips are ready before the
    next step.
  4. Place on ice for 30 sec, spin for 30 sec at room temperature.
  5. Place slide into hybridisation chamber. Add 10 of MilliQ water into
    the wells at each end of the chamber.
  6. Load cooled sample onto array slide and avoid bubles. Carefully
    place coverslip onto slide with fine forceps.
  7. Close and seal lid of hybridisation chamber and submerge in 45oC
    water bath for 14-24 hr.

Desalting the sample for Sep-Pack Purification

Materials:

  • HPLC grade acetonitrile
  • 20 ml ddH2O, 0.22 mm filtered
  • 15 mM TEAA
  • Sep-Pak cartridges, C18 ; 1 column per cy dye. Can be reused twice.
  • 12 mL syringe

Pre-load protocol:

  1. Wash Sep-Pak with 10 mL of acetonitrile.
  2. Wash with 5 mL of water.
  3. Wash with 10 mL of 15 mM TEAA.

Sample Preparation, Loading, and Elution:

  1. The column may be loaded up to 150 A260 units of sample ,
    lyophilized down to 3-5 mL .
  2. Add 1 M TEAA to the sample, to a concentration of 15 mM.
  3. Load the sample onto the column. The dye should be visible near the
    top of the column. Wash the column with 5 mL of 15 mM TEAA.
  4. Elute with 3 mL of acetonitrile. Discard the initial colorless
    drops, collect the colored eluent into a 2 mL centrifuge tube, and
    discard the remaining solution. The collected volume will be
    approximately 1.0-2.0 mL. Some colored material will rema in on the
    column after elution.

Immediately wash and process any subsequent lots, reusing the column
only twice for each lot. If the column is allowed to sit for more than 5
minutes, yields may decrease 5-10% or more. Avoid reusing the column
more than two times , as yields may begin to diminish. Pool the lots
together, check UV absorbance, and calculate yield. Expected yields for
this stage are 70-80% for either Cy dye.

HPLC Preparation

  1. Lyophilize the entire Sep-Pak purified material, keeping the dyes
    separate . Resuspend with a small volume of 0.2 M TEAA.
  2. Centrifuge the sample through a 0.45 mm filter.
  3. Check the absorbance at 260 nm and, if necessary, dilute the sample
    down to a concentration that will not exceed the capacity of the
    column column listed bel ow). Typically a 1 ml injection loop is
    used to inject a 10 mM sample of unlabeled dNTPs + Cy-dUTP.

WASHING

HPLC Purification

Column used: Dionex NucleoPac™ 9 ´ 250 PA-100; P/N 43011 HPLC Equipment:
Perkins Elmer Series 200 LC Pump, Perkins Elmer UV/Vis Detector LC 295

  1. Equilibrate column in buffer system, 3 mL/min flow rate

1.1. 5 min low salt buffer or A1.2. 2 min gradient to 100% high salt
buffer or B1.3. 5 min HSB or B1.4. 2 min gradient to 60% LSB or A1.5. 5
min 60% LSB or A

  1. Load sample into injection loop. See “Final Considerations” on the
    next page for sample lot size and other issues.

  2. Run gradient

3.1. 5 min 60% LSB, 40% HSB3.2. 10 min gradient to 80% HSB3.3. 1 min
gradient to 100% HSB3.4. 10 min 100% HSB3.5. 1 min gradient to 60%
LSB3.6. 5 min 60% LSB

  1. Prepare the following in 2 litres beakersSDS Wash

    990 ml MilliQ water
    10 ml 20x SSC
    2.5 ml 20% SDS

    SSC Rinse

    990 ml MilliQ water
    10 ml 20x SSC
  2. After hybridisation, dry chamber and undo lid. Using flat nosed
    forceps, hold array slide at frosted end and plunge into the SDS
    wash. Move slide sideways until coverslip falls off. Place slide
    into slide holder, and plunge holder for 15 sec, then rock for 3
    min. Plunging every 30 sec or so.

  3. Rapidly transfer slide holder into SSC rinse, and plunge holder for
    15 sec, then rock for 3 min. Plunging every 30 sec or so. SSC will
    start precipitating out if solution starts drying on slide.
  4. Remove slide with forceps and immediately spin slide at 500 rpm for
    5 min at ambient temperature.
  5. Store slides in slide box with closed lid to reduce quenching of
    fluorophores, and scan Cy5 first, as it is more photolabile.

Final Preparative Procedures

For a preparative run of 150 nmoles Cy-dUTP , the recovered fractions of
purified Cy-dUTP is eluted in 5 ml of volatile TEAA buffer that can be
removed by lyophilization.

Lyophilization and Redilution

  1. Pool fractions of each Cy dye into a 50 mL conical tube. Punch holes
    into the cap using an 18-gauge needle. Avoid exceeding more than
    half the volume of the tube, as the samples may have a tendency to
    splatter during degassing or travel up the tube during
    lyophilization.
  2. Freeze the samples on dry ice and transfer them to a lyophilization
    chamber. Wrap the chamber with foil to protect the samples from
    light.
  3. Lyophilize the samples to completion . HPLC-purified samples will
    have the appearance of a residue until most of the TEAA is removed.

    Note: Steps 4 & 5 are for HPLC-purified samples only.

  4. Dilute the samples back up to 8-10 mL with 0.22 mm filtered ddH2O
    and lyophilize until the samples again resemble residue. This time,
    it may require up to 24 hours of lyophilization.

  5. Repeat Step 4. Lyophilization is complete when the sample has the
    appearance of a film and/or specks of solid residue. Resuspend the
    sample in a small volume of 10 mM phosphate buffer, pH 7.0. Bear in
    mind that some of the Cy-dUTP will coat the upp er regions of the
    tube walls and may not be readily visible. Check absorbance of the
    sample. A560/660/A260 ratios should be around 17 for cy 3 and 25 for
    cy 5.

Expected overall yields: cy 3: 50-70%; cy 5: 45-65%.

Final Considerations

Currently 150nmol of Cy-dUTP contained in a 10 mM mixture of labeled and
unlabeled dNTPs is loaded onto the column listed above, depending on the
contents and concentration of the filtrate; PCR flow-through samples
tend to be cleaner and may allow for larger injection lots. Thus, the
flow-through from PCR and RT reactions may be kept separate so that they
can be processed separately. The Sep-Pak purification desalts the sample
and is likely to remove other impurities present in the sample. HPLC
purif ication can still be performed without Sep-Pak purification, but
salt content prohibits injection of more than 50nmol. Furthermore,
yields tend to be lower if the sample is not desalted prior to HPLC
injection.

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