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Note: This protocol is for preparing 16S libraries manually and should only be used to prepare fewer than 96 samples for multiplexing

Materials:
●      Agencourt Ampure XP, A63881 (60mL, $300)
●      2 Roche LichtCycler480 384-well plate
●      1:100 dilution of SYBR stock (Invitrogen S7563, 10,000x)
●      Step 1/ Initial QPCR Primers ( PE_16s_v4U515-F)
●      Step 2 primers ( PE-III-PCR-F, PE-IV-PCR-XXX)
●      Final QPCR primers (BMC Adapter F, BMC Adapter R)
●      HF Phusion (NEB, M0530L)
●      KAPA SYBR 2xMM for final QPCR
●      Invitrogen Super magnet (16 or 8 sample capacity)

Determination of  Step 1 Cycle Time and Sample Check:

Materials used:
○   Contents of MM
○   P200 multi-channel pipette
○    96 well QPCR plate (96 well for opticon stocked in lab)
○   Clear QPCR plate covers

Run this step in duplicate or triplicate to best estimate proper cycling time

Initial QPCR Program (Opticon):
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
52°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Use Ct (bottom of curve, not mid-log) of curves to determine dilutions for step 1 amplification (Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
Breakdown of QPCR amplification math (done to normalize each sample):
○   delta Ct = Sample Ct - highest Ct in sample set (lowest concentration, note: must be Ct of 20 or below)
○   fold = 1.75^(delta Ct)
○   dilution needed = fold
○  note - that input is 2uL per RXN so sample with lowest Ct gets 2uL undiluted
Please note – samples may fail due to too little, too much material, or a poor reaction. It is recommended that failed samples be re-run before moving forward

Library Preparation:
Step 1

Please Note: Samples are run as four 25uL reactions that are pooled at end of cycling

1st step Master Mix 25uL RXN (MM1)

16SStep 1 Program:
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
52°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Run amplification cycle number determined via QPCR (no more than 20 cycles allowed)
After cycling pool duplicates, now have 1x 100uL reaction per sample

SPRI Clean Up
Materials used:
○   SPRI beads
○   70% EtOH
○   EB
○   Invitrogen super magnet
- Vortex cold AmpureXP beads, pool DNA from PCR tubes (~100uL)
- Aliquot 85.5uL beads into Epi’s – let equilibrate to RT
- Add DNA (take 95uL) + beads (85.5uL) = 180.5 uL
- Incubate 13 mins @ RT
- Separate ON magnet 2 mins
- While ON magnet, remove/discard SN
- Wash beads 2x with 70% EtOH, 500uL each wash
- Air dry beads for 15-20mins on magnet
- Remove from magnet, elute in 40uL H2O, vortex to resuspend
- Incubate 7 mins @ RT
-  Separate on magnet 2mins
- Collect 35-40 ul and save SN

Sample Re-Aliquoting and Step 2
Please Note: Samples are run as four 25uL reactions that are pooled at end of cycling

16s Step 2 Program:

Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
83°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Run 7 cycles of amplification
- After cycling pool duplicates, now have 1x 100uL reaction per sample

SPRI Clean Up
Materials used:
○   SPRI beads
○   70% EtOH
○   EB
○   Invitrogen super magnet
- Vortex cold AmpureXP beads, pool DNA from PCR tubes (~100uL)
- Aliquot 85.5uL beads into Epi’s – let equilibrate to RT
- Add DNA (take 95uL) + beads (85.5uL) = 180.5 uL
- Incubate 13mins @ RT
- Separate ON magnet 2mins
- While ON magnet, remove/discard SN
- Wash beads 2x with 70% EtOH, 500uL each wash
- Air dry beads for 15-20mins on magnet
- Remove from magnet, elute in 40uL H2O, vortex to resuspend
- Incubate 7mins @ RT
-  Separate on magnet 2mins
- Collect 35-40 ul and save SN

Final QPCR
Once you have a substantial or all of your samples prepared you can run a final QPCR to determine dilutions and volumes for multiplexing. This step also confirms that the library preparation was successful

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Activation:
95°C - 5 minutes
Amplification:
95°C – 30 seconds
60°C – 45 seconds (1-step annealing/extension)

Run 35 cycles of amplification

Use mid-log phase of curves to determine volumes for multiplexing (Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
Please note – samples may fail due to too little, too much material, or a poor reaction. It is recommended that failed samples be re-run before moving forward
Breakdown of QPCR multiplexing math (done to normalize each sample):
○  delta Ct = Sample Ct - highest Ct in sample set (lowest concentration)
○  fold = 1.75^(delta Ct)
○  ratio = 1/fold
○  volume to mix b/c of ratio = X*ratio (X = minimum desired volume per sample)
○  how to dilute = fold
○  note - sample with lowest Ct will get an undiluted Xuls added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples

Sample Multiplexing and Submission for Sequencing:
- Once samples have been multiplexed aliquot ~20uL of the final mix and submit it to the BioMicro Center for sequencing