Tecan array protocol

2015-01-13

azim58 - Tecan array protocol

Original Protocol: SOP for Binding Antibodies to the CIM10K_121709.doc

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Tecan Array Protocol

* Materials
  o Tecan Array Instrument (Tecan HS 4800 Pro which is a microarray
    hybridization station)
  o Blocking Buffer (Bart mentioned this should be prepared fresh
    about every month or even 2 weeks)
    - Super G buffer for nitrocellulose slides
    - BSA based buffer most peptide based slides
      @ 5 mL of 30% BSA (or 1.5 g BSA and 5 mL H20 so 45 mL H20
        instead of 40 mL H20)
        # last seen 4 C fridge by -20 freezers

@ 6.9 uL Mercaptohexanol
        # last seen above Zbig's bench

@ 25 uL of Tween 20
        # last seen above scales

@ 5 mL 10X PBS
      @ 40 mL ddH20

o Incubation buffer (Bart mentioned this should be prepared fresh
    about every month or even 2 weeks)
    - BSA based buffer for most peptide slides
      @ 5 mL of 30% BSA (or 1.5 g BSA and 5 mL H20 so 45 mL H20
        instead of 40 mL H20)
      @ 25 uL of Tween 20
      @ 5 mL 10X PBS
      @ 40 mL ddH20

- Note that this incubation buffer can also be used after Super
      G blocking buffer on the nitrocellulose slides

* General Tecan Instrument Protocol (this is just a link for reference
  to the type of protocol on the computer used for incubating and washing
  the slides)
*

* Certain types of slides (like aminosilane slides) should be washed
  with post print wash buffer before use in the tecan. Post print wash
  protocol. Note washing nitrocellulose slides with H20 alone seems to
  work just fine.

* Prepare appropriate dilutions of samples. You may want to do this
  during the first blocking step when the Tecan is up and running.
  o Necessary volumes
    - Samples for one gasket chambers: 200 uL (might be best to add
      a little extra like 210 uL)
    - Samples for two gasket chambers: 100 uL (might be best to add
      a little extra like 110 uL or 105 uL + 5 uL incubation buffer for dye)
    - Total volume (# samples)*(1 or 2 depending on gasket chamber
      type)*(volume for gasket)
      @ Example: 4*2*125*uL = 1000*uL

o Molarity of Antibody from mg/mL (you may want know this when
    diluting from commercial stocks)
    - 2*mg/(1*mL)*1000*mL/(1*L)*1*g/(1000*mg)*(1*mol)/(150000*g)*1*10
      ^9*nmol/(1*mol) = 13333*nmol/L

o Primary sample
    - Sera: people usually use a 1:500 dilution.
      @ Ex. 1/500 = y/600 -> y = 1/500*600*uL = 1.2 uL in 600 uL

o Biotinylated Secondary
    - usually 5 nM is used
      @ note that for the nitrocellulose cell lysate slides 5 nM
        appears to be too much. 0.1 nM may be the better concentration to
        use.

- Ex. 667*nM*y/(600*uL)=5*nM -> y = 5*nM*600*uL/(667*nM) = 4.5
      uL in 600 uL
    - Dye with biotinylated secondary (0.1 nM)
      @ The landing lights on most arrays is a biotinylated
        peptide. Therefore, in order to detect the landing lights, it is
        necessary to use streptavidin AlexaFluor. If direct labeled
        secondary is used, the landing lights will not be visible unless
        a small amount (such as 0.1 nM) of streptavidin AlexaFluor is
        added as well.

o Dye (streptavidin labelled)
    - AF647 1300 g/mol, streptavidin 60,000 g/mol
    - Ex 2
      mg/mL*1*g/(1000*mg)*1*mol/(1300*g+60000*g)*1000*mL/(1*L)*1*10^9*nmol/
      (1*mol) = 32626*nM -> Can dilute 1 to 100 to get 326.3 nM
      streptavidin AF647
    - Ex. 370*nM*y/(600*uL)=5*nM -> y = 5*nM*600*uL/(370*nM) = 8.1
      uL in 600 uL

* Load chambers into the chamber frame (it may be best to make sure
  chambers are fully washed and dried!).
  o If the seals on the gasket chambers are too old then liquid will
    leak in the chambers, and then the slides are likely to break when
    the chambers are opened after the binding experiment.
  o May want to use dry chambers and spray out holes at bottom of
    chamber to clear out any remaining fluid in the microfluidic tubes.
  o I'm not sure if chambers need to be loaded into blocks that are
    not used. Rebecca said she doesn't think so.

* Fill fluid reservoirs with appropriate buffer and ensure that the
  waste container is empty.
  o Bottles one and two are TBST and bottle five is ddH20.
  o Note that the liquid in these bottles only decreases by a few
    centimeters in a run. In one run, bottle 1 decreased 2.6 cm, bottle 2
    decreased 1 cm, and bottle 6 decreased 0.8 cm.

* Turn on the nitrogen supply. The second regulator stage should be no
  more than 45 PSI or the safety valve will be triggered. Do not remove
  or inhibit the safety valve. If the first regulator stage is below 500
  PSI, there will not be enough nitrogen to complete the run and the the
  nitrogen cylinder will need to be exchanged.
* Attach tubes to fluid reservoirs and run the prime function for
  channel one.
* May want to clear off platform surface by blowing nitrogen air across
  the surface.
* Place slides in the black slide holder (slide adaptor) and load onto
  stage. Lift black slide holder along with slides out of instrument and
  press down on edge of slides to make sure they are all the way down.
  Some people also think that if the slides are pushed more to the right
  side than the left that the chamber frame will close better. Ensure
  that the slides are properly loaded so they are not broken by the
  silver clips on the chambers. Blank slides should be placed in empty
  slots. You may want to record the position of the slides.
* Gently close the chamber frame. Screw in black plugs. Stop and
  reposition slides if a scratching sound is heard.
* Load program. The necessary program to run will depend on how many
  hybridizations will be used.
  o Common programs used are CIM Immunosignature SOP and CIM
    Immunosignature SOP three step as well as Overnight primarysecondary
    only Tecan Protocol. If using nitrocellulose slides, it may be a good
    idea to add another wash before the primary step.

* Start the program (not the prime or the rinse or anything else). Read
  the on-screen checklist and ensure that all are completed.
  o The initial rinse takes about 2 min 30 s.

* Add reagents as the program runs
  o Apply each sample type in order. Note that after the dye is
    added, 10 uL of incubation buffer should also be added after it, and
    the light shield should be placed over the chamber. Note that the
    slides will automatically be dried at the end of the run.
    - Blocking buffer (load the blocking buffer)
      @ Make sure to use Super G blocking buffer for
        nitrocellulose slides
      @ Note that you may want to prepare the dilutions of
        samples after the blocking buffer has been added if this has not
        already been done.

- Primary sample (load primary sample)
    - Secondary (load secondary). If the dye was added, follow the
      instructions above (may want to start warming up the lasers on the
      scanner at this point).
      @ The landing lights on most arrays is a biotinylated
        peptide. Therefore, in order to detect the landing lights, it is
        necessary to use streptavidin AlexaFluor. If direct labeled
        secondary is used, the landing lights will not be visible unless
        a small amount (such as 0.1 nM) of streptavidin AlexaFluor is
        added as well.

- Tertiary (load tertiary) (not all runs have a tertiary. e.g.
      if the secondary already has an attached dye). If the dye was
      added, follow the instructions above (may want to start warming up
      the lasers on the scanner at this point).

o Loading a sample
    - When prompted by the program, for the chamber indicated by
      the + sign on the instrument screen, unscrew the black plug.
    - Using a P200, place the tip down to the bottom of the sample
      injection port and slowly depress plunger to the first stop. Do not
      depress to the second stop or you will introduce bubbles which will
      negatively impact the resulting data.
    - Replace the black plug. Note that if the black plug is not
      screwed on tight enough, then some liquid may be sucked out of the
      chamber. Don't screw on the black plug too tight though.
    - Press ok on the instrument and similarly load remaining
      samples

o After the protocol is finished, the machine will take about 16
    min for the final slide drying.

* When the program is finished, open the chamber frame carefully. Press
  down with your fingers and lift up on the latch with your thumbs to
  avoid breaking slides. Also, don't lift the chambers right away. Wait
  20-30 seconds for everything to depressurize, and then lift up to avoid
  the slides sticking to the chambers.
* If the gaskets have failed and evidence of leakage can be seen, wash
  the slides 1X in TBST and 2X ddH20 using the manual wash method (slide
  in slide holder dipped back and forth in cube container filled with
  liquid for 30 s). Centrifuge dry at 1500 rpm for 5 min.
* Scan slides at appropriate wavelength with 100% PMT on the Agilent
  scanner
  o 555 nm is for green light
  o 647 nm is for red light
  o Grace Biolabs nitrocellulose slides scan best on the Tecan Power
    Scanner (see Josh Lebaer Lab Scanner). Scan takes about 9 min/slide @
    10 um resolution.
	-be careful not to let the scanner lid slam shut.  It will just fall down if it is not pushed all the way back.

* Shutdown the instrument
  o Run a rinse program with final system drying before shutting down
    for the day. The computer will provide more instructions once the
    rinse button is clicked.
    - Note that the tubes are submerged in H20 for about 21 min
    - The tubes dry outside of water for about 11 min.
    - If nitrocellulose slides were used, make sure there is not a
      white residue on the chambers or the glass slides leftover. If
      there is a white residue, the glass slides can be discarded, and
      the rinse can be repeated with new glass slides. It may be best to
      wash the instrument twice regardless.

o Remove the chambers from the chamber frame. Remove the black
    sample port plug and soak the chambers and plugs in distilled water
    for about 30 min to an hour or longer (Bart likes to set them down on their side like Lincoln logs so that main surface does not get scratched). After the soak, it may be a
    good idea to blow out the water with the nitrogen nozzle at the holes
    in the chambers, and then lay them out on paper towels to dry.
  o Empty the waste container (probably on the floor below
    instrument) if it looks fairly full.
  o The chamber frames should be left resting on the locking pins and
    not left closed.
  o Note that the original protocol mentions that the nitrogen tanks
    should be turned off. However, I have never observed anyone but
    myself doing this, and it seems to make things more inconvenient for
    the next person that uses the Tecan. Therefore, it's probably best to
    leave the nitrogen tanks on.

* Remember to take slides out of scanner once the data is obtained.

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