Page Comparison

*This is what I would do to QC for an entire library prep.

MasterMix (make for 84 plates in 2x 50mL conical tube)

1. Take out 13 tubes of dNTPs and 17 tubes of 5x Buffer to thaw.

2. Get six new 50 ml tubes from pre-PCR side and fill up with Ultra Pure Water from the EcoBGC. Label tubes 1-6.

3. Using the 25mL combitip, aliquot 25mL and then the remaining 6,276.5mL into a new pre-PCR side 50mL tube. Repeat again into a second new pre-PCR side 50mL tube.

4. Once thawed, combine all 17 tubes of 5x Buffer into a 50mL tube labelled 5x KAPA Hifi Buffer

5. Once thawed, combine all 13 tubes of dNTPs into a 5mL tube labelled 10mM dNTP

6. Combine 11 tubes of KAPA Hifi HS DNA Pol into one 5mL tube labelled KAPA Hifi HS DNA Pol

7. Add the remaining master mix reagents to each tube in the quantities listed below using appropriate combitips or pipettes.

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MMX2 NOTE: Master-mix should not go through more than three freeze-thaws. If using a reservoir, poor any leftover mastermix into a new labelled tube and mark with a tally for freeze-thaws if mastermix was previously frozen. If using a combitip, close mastermix tube and mark with a tally to keep track of freeze thaws. If mastermix is being QC’d immediately after being made, no tally is needed until the master mix has been frozen.

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MagBead Cleanup Columns 1, 3, & 5

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:

• Equilibrate Beads to room Temperature

• Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

• Pipette mix up and down 10 times.

• Incubate at RT for 5 minutes

• Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

• Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

• Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

• Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

• Reaspirate from each well to assure maximum EtOH removal

• Allow plate to air dry for 7 minutes.

• Remove sample plate from magnet plate.

• Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.

• Place sample plate back on magnet for 5 minutes or until all wells are cleared.

• Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

• Store in -20 until ready to qPCR.

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• Add 16 ul of Illumina Library Quantification MasterMix to each well:

• Add 4 ul of template, pool, or standards to each well:

Results:

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• If QC passes, aliquot all remaining UPW into 2mL tubes and label with UPW# and aliquot date.

• If QC passes, aliquot TE bottles into a variety of 5mL, 15mL, and 50mL tubes and label with bottle number, lot number, and aliquot date.

• You could aliquot the MagBeads like the TE as well unless it is not advised to aliquot out that reagent. TBD.

NOTE: You can scale this back and only QC TE, UPW, and MagBeads just using the first MMX instead of both. You can just QC MMX2 using TE or one UPW tube as the sample, and skip the MagBead for MMX2 as well and just MagBead 1 & 3. This would allow you to run just two columns for MMX2 for 16S and ITS, allowing for 6 lanes instead of 8. I honestly would probably do that instead but just in case you were wanting to look at comparability data between master mixes I just made them the same.

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