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• This protocol is for preparing 16S libraries on Rosie (the Alm lab's epMotion 5075)  and should only be used to prepare 96 well sample plates for multiplexing. 
• At the beginning of EACH DAY using Rosie spray and wipe down the deck with RNAse AWAY and 70% EtOH then UV the deck for 10-15minutes before starting protocols for the day.
• When starting the protocol, turn on the computer first, THEN Rosie.  When finished, turn Rosie off first, followed by the computer.
• If you are preparing only one plate for sequencing please use the PE_16s_V4_U515_F primer in the first step PCR. If you are planning on combining multiple 96 well plates for sequencing (4 or more recommended) please use the PE_16s_V4_F_Bar## primer for your first step PCR. 

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Rosie Protocol Used: '16s Stp1 384 QPCR.dws', please note that this protocol can be run for 1 or 2 sample plates at once (there is a promt prompt half way through the protocol that will ask if you are running tow two plates)

Materials used:

• Contents of MM
• 384 well Lichtcycler Lightcycler QPCR plate
• Clear PCR plate covers
• 50uL filtered epTIPS x 2
• 300uL non-filtered epTIPS x 1
• 30mL reagent reservoir x 1

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Initial QPCR Program (Roche Lighcycler Lightcycler 480):
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 - lowest highest Ct in sample set
○   fold = 1.75^(delta Ct)
○   dilution needed = fold
○  note - that input is 2uL per RXN so sample with lowest highest 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

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Materials used:

• SPRI beads (8.5mL9mL)
• 70% EtOH (30.2ml32mL)
• EB (4.1mL5mL)
• 96 well magnet plate
• 3 30mL reagent reservoirs
• 1 100mL reagent reservoir (for waste)
• 1 Axygen 96 well skirted PCR plate
• 3 boxes of 300uL filtered epTIPS
• 5 boxes of 300uL un-filtered epTIPS

Do not put cover on EtOH reservoir tightly after filling, the volume will be too high to do this with out spilling.

Note: At one point during the protocol, Rosie will ask the user to replace empty tip boxes.  Replace the boxes in B1 and B2 with new boxes.  When the protocol is restarted, it will prompt you to take action on the empty tip box in B4.  Choose “ignore” and the program will continue without problems.

Step 2 (one plate at a time):

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Materials used:

• SPRI beads (8.5mL9mL)
• 70% EtOH (30.2ml32mL)
• EB (4.1mL5mL)
• 96 well magnet plate
• 3 30mL reagent reservoirs
• 1 100mL reagent reservoir (for waste)
• 1 Axygen 96 well skirted PCR plate
• 3 boxes of 300uL filtered epTIPS
• 5 boxes of 300uL un-filtered epTIPS

Do not put cover on EtOH reservoir tightly after filling, the volume will be too high to do this with out spilling.

Note: At one point during the protocol, Rosie will ask the user to replace empty tip boxes.  Replace the boxes in B1 and B2 with new boxes.  When the protocol is restarted, it will prompt you to take action on the empty tip box in B4.  Choose “ignore” and the program will continue without problems.

Final QPCR (this is a copy of the BMC QPCR for quality control, it can be modified to use normal SYBR green as well):
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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Final QPCR Program (Opticon or LichtcyclerLightcycler)

Activation:
95°C - 5 minutes
Amplification:
95°C – 30 seconds
60°C – 45 seconds

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Use mid-log phase of curves to determine volumes for multiplexing (use 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 - lowest highest Ct in sample set
○  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 highest Ct will get an undiluted Xuls XuLs added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples

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Final QPCR Program (Opticon or LichtcyclerLightcycler)

Activation:
95°C - 5 minutes
Amplification:
95°C – 30 seconds
60°C – 45 seconds

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Use mid-log phase of curves to determine volumes for multiplexing (use 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 - lowest highest Ct in sample set
○  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 highest Ct will get an undiluted Xuls XuLs added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples

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