Dinh/Dinh 2016/NOTES/2016-8-24

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  1. Thaw the MiSeq reagent cartridge from -20 C (in gene sequencing room) in a bath of warm water first. The HT1 buffer is included in the reagent cartridge box and needs to be thawed as well (helps to use a block to hold the HT1 buffer tube in the warm water)
  2. Pool all the samples together and find out the final concentration in nM.
  3. Fill out the MiSeq excel denature and pool spreadsheet File:Miseq denature and pool.xlsx
    1. File:Derek MiSeq Spreadsheet.jpg
  4. Enter the sample concentration on the spreadsheet and the volumes (in uL) of Qiagen Elution Buffer, sample, and HP3 (2N NaOH) will be automatically calculated. These calculations assume a desired final concentration of 20 pM. If you want to load a higher concentration, double the volumes listed on the spreadsheet (will result in a total reaction volume of 20 uL instead of 10 uL).
  5. The denature/pool components which can be found in the 4C fridge (Derek's box w/ yellow tape): Qiagen Elution Buffer, HP3 (2N NaOH), and 200 mM Tris
    1. File:Derek MiSeq yellowbox.jpg
    2. File:Derek MiSeq 3components.jpg
  6. Get a new 1.5 mL tube and label with an X on the top and add in this order: Qiagen Elution Buffer, pooled sample, and then HP3. This denatures the dsDNA.
    1. File:Derek MiSeq labelX.jpg
    2. Example: for an 18 nM pooled sample: add 17.89 uL of Qiagen elution buffer, 1.11 uL of pooled sample, and 1 uL of HP3,
  7. Vortex the tube for 20 seconds & spin down.
  8. The reaction needs to run for at least 5 minutes so start a timer.
  9. While waiting, prepare the flowcell (from 4C fridge).
    1. Take the flowcell out of its container & rinse quickly with Milli-Q water. Gently dab dry with a KimWipe. Spray a KimWipe with 75% ethanol and then gently wipe the flowcell on both sides.
    2. File:Derek MiSeq rinseFlowcell.jpg
    3. Flowcell needs to be dried completely.
    4. File:Derek MiSeq dryCompletely.jpg
    5. Open up the tab and blot gently to completely dry the flowcell then snap the cover back on using its hinge.
    6. File:Derek MiSeq flowCellHinge.jpg
  10. Load the flowcell into the MiSeq instrument.
    1. From the home page, go to Sequence, then log into your BaseSpace account.
    2. Open the left black compartment, press the white button to release the flowcell. Discard old flowcell, gently place new flow cell in and close everything.
    3. File:Derek MiSeq insertFlowCell.jpg
    4. On the instrument screen there should be a green check but there is a glitch in our system so we need to press back and re-login again. There should be a green check mark after the second login
    5. Click Next
  11. Open the right black compartment, check the waste container (the white-ish container on the right side), every run adds about 1.5 in of waste, so make sure there's enough room.
    1. Change the incorporation buffer (PR2) to the new one from the box with the flowcell.
    2. There should be a green check and you should be able to click Next.
  12. Get back to the denaturing sample, make sure at least 5 minutes have passed.
    1. File:Derek MiSeq fiveMinutes.jpg
  13. Neutralize by adding an equal volume of 200 mM Tris (10 uL if you used the standard volumes in the denaturing reaction, 20 uL if you doubled the volumes). Pipette up and down then vortex and spin down
  14. Dilute to pM with HT1:
    1. HT1 buffer is found attached to the MiSeq kit tray.
    2. File:Derek MiSeq HT1.jpg
    3. Vortex it for 3-6 seconds after it had been thawed.
    4. Get a new 1.5 mL tube and label with "LOAD"
    5. Example: to get to 30 pM, add 564 uL of HT1 to 36 uL of combined sample.
    6. To spike in PhiX, mix diluted sample with 20 pM denatured PhiX in the desired ratio. For example, for a 1% PhiX spike in mix 594 uL diluted sample with 6 uL 20 pM PhiX.
  15. CUSTOM primer addition is next! For standard primers (HP10, HP11, etc.) no need to change any of the ports
    1. Invert reagent cartridge a few time after it's been thawed!!!
    2. NOTE: Port 12 - Read 1 primer, Port 13 - Index primer, Port 14 - Read 2 primer. Derek has a guide posted at his bench.
    3. File:Derek MiSeq trayPorts.jpg
    4. Puncture the appropriate port with a 1000 uL tip.
    5. Get a new tube and label with the custom primer and port.
    6. Get a 1 or 2 mL serological pipette tip and aspirate the reagent inside the port (should be about 600 uL) then transfer the reagent to the newly labeled tube. A sero pipette is necessary because the port is too narrow for a 1000 uL pipette.
    7. Get the custom primer out from the Sequencing primers box (in the 4C) and add 3.4 uL of 100 uM primer. Up to 10 uL of primer may be added but 3.4 uL should be more than enough for most cases.
  16. Vortex "LOAD" tube and all custom primer tubes for 20 s.
  17. Spin the tubes down with centrifuge at max speed for 1 min.
  18. Prepare the sample spreadsheet using the Illumina Experiment Manager. The software is installed on the computer next to the qPCR.
    1. Make sure to check Fastq-Only
    2. Change the barcode to today's date, Experiment name is also today's date with the experimenter's names
    3. Make sure the cycle numbers are correct. (Other fields are not necessary)
    4. Enter sample IDs and indices. (Other fields are not necessary)
    5. You can email it to yourself to transfer the spreadsheet to the MiSeq device
    6. File:Derek MiSeq experimentManager.jpg
  19. Get the "LOAD" tube, and custom primer tubes.
    1. Puncture the "LOAD" port on the MiSeq reagent cartridge
    2. Dispense "LOAD" tube and primer tubes into the MiSeq reagent cartridge
    3. File:Derek MiSeq loading.jpg
    4. Load the cartridge into the cooler compartment on the instrument.
  20. Click "Change sample spreadsheet" and load the sample spreadsheet you emailed yourself
  21. Check run information. Proceed through clicking Next.
    1. File:Derek MiSeq checking.jpg
  22. Wait for flow rate checking to complete ~approx. 3 minutes.
  23. Click the blue start run button & its' good to go.
    1. File:Derek MiSeq goodtogo.jpg

Washing after every run[edit]

  • The wash tray is a gray tray stored in the 4 C resembling the reagent cartridge in shape
  • Fill each wash tray port with wash buffer (stored next to the MiSeq) using a serological pipette
  • Take out reagent cartridge (if still in MiSeq) and replace with wash tray
  • There's wash buffer in a PR2 incorporation buffer bottle (should be labeled). Place this bottle where the PR2 incorporation buffer normally goes.
  • Hit start wash

Washing when instrument maintenance is required[edit]

  • Repeat washing step 3X.