*This is what I would do to QC for an entire library prep.
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Aliquot 4500 ul into ten 5ml tubes and the remaining 2,454uL into an additional 5mL tube. Label 16S/ITSAmpStd_MMX1_Prep-Date*_NS#** and 16S/ITSAmpStd_MMX2_Prep-Date_NS#.
*Prep-Date is the day the mastermix is prepped, i.e. 06/17/22
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| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | MMX1 + 2uL 16S + 2uL TE Bottle 1 | MMX1 + 2uL 16S + 2uL TE Bottle 1 | MMX1 + 2uL ITS + 2uL TE Bottle 1 | MMX1 + 2uL ITS + 2uL TE Bottle 1 | MMX2 + 2uL 16S + 2uL TE Bottle 1 | MMX2 + 2uL 16S + 2uL TE Bottle 1 | MMX2 + 2uL ITS + 2uL TE Bottle 1 | MMX2 + 2uL ITS + 2uL TE Bottle 1 | NTC | NTC | NTC | |
B | MMX1 + 2uL 16S + 2uL TE Bottle 2 | MMX1 + 2uL 16S + 2uL TE Bottle 2 | MMX1 + 2uL ITS + 2uL TE Bottle 2 | MMX1 + 2uL ITS + 2uL TE Bottle 2 | MMX2 + 2uL 16S + 2uL TE Bottle 2 | MMX2 + 2uL 16S + 2uL TE Bottle 2 | MMX2 + 2uL ITS + 2uL TE Bottle 2 | MMX2 + 2uL ITS + 2uL TE Bottle 2 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |
C | MMX1 + 2uL 16S + 2uL UPW 3 | MMX1 + 2uL 16S + 2uL UPW 3 | MMX1 + 2uL ITS + 2uL UPW 3 | MMX1 + 2uL ITS + 2uL UPW 3 | MMX2 + 2uL 16S + 2uL UPW 3 | MMX2 + 2uL 16S + 2uL UPW 3 | MMX2 + 2uL ITS + 2uL UPW 3 | MMX2 + 2uL ITS + 2uL UPW 3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |
D | MMX1 + 2uL 16S + 2uL UPW 4 | MMX1 + 2uL 16S + 2uL UPW 4 | MMX1 + 2uL ITS + 2uL UPW 4 | MMX1 + 2uL ITS + 2uL UPW 4 | MMX2 + 2uL 16S + 2uL UPW 4 | MMX2 + 2uL 16S + 2uL UPW 4 | MMX2 + 2uL ITS + 2uL UPW 4 | MMX2 + 2uL ITS + 2uL UPW 4 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |
E | MMX1 + 2uL 16S + 2uL UPW 5 | MMX1 + 2uL 16S + 2uL UPW 5 | MMX1 + 2uL ITS + 2uL UPW 5 | MMX1 + 2uL ITS + 2uL UPW 5 | MMX2 + 2uL 16S + 2uL UPW 5 | MMX2 + 2uL 16S + 2uL UPW 5 | MMX2 + 2uL ITS + 2uL UPW 5 | MMX2 + 2uL ITS + 2uL UPW 5 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |
F | MMX1 + 2uL 16S + 2uL UPW 6 | MMX1 + 2uL 16S + 2uL UPW 6 | MMX1 + 2uL ITS + 2uL UPW6 | MMX1 + 2uL ITS + 2uL UPW6 | MMX2 + 2uL 16S + 2uL UPW 6 | MMX2 + 2uL 16S + 2uL UPW 6 | MMX2 + 2uL ITS + 2uL UPW6 | MMX2 + 2uL ITS + 2uL UPW6 | 2 pM Std | 2 pM Std | 2 pM Std | |
G | MMX1 + 2uL 16S + 2uL MC | MMX1 + 2uL 16S + 2uL MC | MMX1 + 2uL ITS + 2uL MC | MMX1 + 2uL ITS + 2uL MC | MMX2 + 2uL 16S + 2uL MC | MMX2 + 2uL 16S + 2uL MC | MMX2 + 2uL ITS + 2uL MC | MMX2 + 2uL ITS + 2uL MC | 20 pM Std | 20 pM Std | 20 pM Std | |
H | MMX1 + 2uL 16S + 2uL MC | MMX1 + 2uL 16S + 2uL MC | MMX1 + 2uL ITS + 2uL MC | MMX1 + 2uL ITS + 2uL MC | MMX2 + 2uL 16S + 2uL MC | MMX2 + 2uL 16S + 2uL MC | MMX2 + 2uL ITS + 2uL MC | MMX2 + 2uL ITS + 2uL MC |
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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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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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