Page History

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• 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; NOTE - PE-IV-PCR-XXX primers should be used as a pre-pipetted 96well plate)
• Final QPCR primers (PE Seq F, PE Seq R)
• HF Phusion (NEB, M0530L)
• KAPA SYBR 2xMM for final QPCR
• Invitrogen Super magnet (16 or 8 sample capacity)
• 96 well magnet plate
• X boxes of 300uL filtered epTIPS (Cat#XX)
• X boxes of 300uL unfiltered epTIPS (Cat#XX)
• X boxes of 50uL filtere epTIPS (Cat#XX)

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• 50uL filtered epTIPS x 2
• 30mL reagent reservoir x 1
• EB
• 1 Axygen 96 well skirted PCR plate

Library Preparation:

Step 1 (one plate at a time)

Rosie Protocol Used: '16s Step 1 PCR.dws'

Materials used:

• 4 Axygen 96 well skirted PCR plates
• 1 row of a 300uL non-filtered epTIPS box
• 1 box of 50uL filtered epTIPS
• 1 30mL reagent reservoir
• Clear PCR plate covers

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

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16SStep 1 Cycling 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:

Rosie Protocol Used: '4x to 96pool.dws'

Materials used:

• 1 Axygen 96 well skirted PCR plate
• 1 box of 300uL filtered epTIPS

96 well SPRI Clean Up

Rosie Protocol Used: 'SingleSPRI.dws'

Materials used: ○   

• SPRI beads (8.5mL)

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• 70% EtOH (30.2ml)

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• EB (4.1mL)
• 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

Step 2 (one plate at a time):

Rosie Protocol Used: '16s Step2 PCR.dws'

Materials used:

• 4 Axygen 96 well skirted PCR plates
• 1 pre-pipetted PE-PCR-IV-XXX barcode plate
• 3 boxes 50uL filtered epTIPS
• 1 30mL reagent reservoir
• Clear PCR plate covers

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

16s Step 2 Cycling 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:

Rosie Protocol Used: '4x to 96pool.dws'

Materials used:

• 1 Axygen 96 well skirted PCR plate
• 1 box of 300uL filtered epTIPS

** Now you have a choice!***

If your sample plate is a mixture of sample types (stool and saliva for example) or you have reason to suspect something went wrong:

96 well SPRI Clean Up

Rosie Protocol Used: 'SingleSPRI.dws'

Materials used: ○   

• SPRI beads (8.5mL)

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• 70% EtOH (30.2ml)

...

• EB (4.1mL)
• 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

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!

Final QPCR Program (Opticon or Lichtcycler)

Heat:
95°C – 5 minutes
Amplify:
95°C – 10 seconds
60°C – 20 seconds
72°C – 30 seconds
Melting Curve:
95°C – 5 seconds
65°C – 1 minute
97°C - continuous
Cool:
40°C – 10 seconds
Run 35 cycles of amplification
Use mid-log phase of curves to determine volumes for multiplexing (use Illumina Library QPCR and Multiplexing document)
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 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 Ct will get an undiluted Xuls added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples

SPRI Clean Up
○   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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