Tecan+array+protocol

Original Protocol:

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Tecan Array Protocol >
 * Materials
 * Tecan Array Instrument (Tecan HS 4800 Pro which is a microarray hybridization station)
 * 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
 * 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.
 * 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.
 * 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)
 * Total volume (# samples)*(1 or 2 depending on gasket chamber type)*(volume for gasket)
 * Example: 4*2*125*uL = 1000*uL
 * 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
 * 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
 * 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.
 * 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!).
 * 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.
 * May want to use dry chambers and spray out holes at bottom of chamber to clear out any remaining fluid in the microfluidic tubes.
 * 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.
 * Bottles one and two are TBST and bottle five is ddH20.
 * 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.
 * 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.
 * The initial rinse takes about 2 min 30 s.
 * Add reagents as the program runs
 * 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).
 * 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 than 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
 * 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
 * 555 nm is for green light
 * 647 nm is for red light
 * 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.
 * Shutdown the instrument
 * 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.
 * 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. 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.
 * Empty the waste container (probably on the floor below instrument) if it looks fairly full.
 * The chamber frames should be left resting on the locking pins and not left closed.
 * 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.