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Pool Cleanups for 1BUN (PJD), 1SMIN, Trout1_2, and Trout3_4

Pool Cleanups for 1BUN (PJD), 1SMIN, Trout1_2, and Trout3_4

@Shannon Harris

  • Combine 3 ul from each well of the final PCR plates for 1BUN2 (16 samples) and 1BUN2 (28 samples including the few from Josh)

  • Combine 3 ul into a separate pool from each well of the 1SMIN1+1SMIN2 final PCR plate (96 samples)

  • If there is 100 ul or more left each of non size selected Trout1_2 and Trout3_4, use 100 ul of each.

    • If not adjust to bring to 100 ul (Added ________ ul)

  • Add 100 ul of each of these to separate wells of the Nucleofast Cleanup plate

 

1

2

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12

 

1

2

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12

A

 

 

 

 

 

 

 

 

 

 

 

 

B

 

 

C

 

 

D

 

 

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G

 

 

H

 

 

  • Place Nucleofast Plate on vaccum manifold. Ensure vent is closed on manifold. Turn vacuum on. Adjust right side control if vacuum outside -0.4 to -0.6 bar (-400 to -600 mbar) Leave vacuum on 10-15 minutes or 30 - 60s after liquid has passed through membrane

  • Add 100uL of UPW to each well to clean up per the protocol instructions. Leave vacuum on until all the UPW has passed through the membrane.

  • Release vacuum 60-90s by turning off vacuum and releasing vent on manifold

  • Dispense 100 ul TE for all wells (Trout, 1Smin, 1Bun)

  • Incubate at RT for 5 minutes

  • Pipette up and down half the lowest V of TE 10 times to mix each well, then transfer to labeled tubes.

  • Size select the trout samples on the Pippin Prep (2 of each if there is enough V)

  • Run the size selected Trout and cleaned bird samples on a Tape Station at INBRE.

Part 2

  • INBRE Tapestation results showed that the clean up worked to get the samples into the correct range but the concentration is now too low. We will do the following to increase concentration.

    • Add _________ (increased volume) of uncleaned and not size selected Trout Pool 1_2 and Trout Pool 3_4 to wells on the Nucleofast Clean up plate.

    • Place Nucleofast Plate on vaccum manifold. Ensure vent is closed on manifold. Turn vacuum on. Adjust right side control if vacuum outside -0.4 to -0.6 bar (-400 to -600 mbar) Leave vacuum on 10-15 minutes or 30 - 60s after liquid has passed through membrane

    • DO NOT DO CLEAN UP STEP

    • Release vacuum 60-90s by turning off vacuum and releasing vent on manifold

    • Dispense 100 ul TE for all wells

    • Incubate at RT for 5 minutes

    • Pipette up and down half the lowest V of TE 10 times to mix each well, then transfer to labeled tubes.

    • Size select the trout samples on the Pippin Prep (2 of each if there is enough V)

Pool pools

  • 192 samples in SMIN pool and 104 samples in BUN pool for 296 total

  • Size select 2 times at 275-375 bp on Pippin Prep

  • Run on High Sensitivity Tapestation

  • Combine 48 ul SMIN and 26 ul BUN for 74 ul total for equal volumetric pooling per sample