16S by hand library prep

16S By Hand Library Preparation

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 OR PE_16s_v4_Bar#,  PE_16s_v4_E786_R)

●      Step 2 primers ( PE-III-PCR-F, PE-IV-PCR-XXX)

●      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)

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

Initial QPCR master mix (MM)

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 (google docs, Illumina Library QPCR and Multiplexing)

Breakdown of QPCR amplification math (done to normalize each sample):

○   delta Ct = Sample Ct - lowest Ct in sample set

○   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)

16s Step 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’ @ RT

- Separate ON magnet 2’

- While ON magnet, remove/discard SN

- Wash beads 2x with 70% EtOH, 500uL each wash

- Air dry beads for 15-20’ on magnet

- Remove from magnet, elute in 40uL H2O, vortex to resuspend

- Incubate (at least 7’)

-  Separate on magnet 2’

- 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

2nd step Master Mix  25uL RXNs (MM2)

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 9 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 13’ @ RT

- Separate ON magnet 2’

- While ON magnet, remove/discard SN

- Wash beads 2x with 70% EtOH, 500uL each wash

- Air dry beads for 15-20’ on magnet

- Remove from magnet, elute in 40uL H2O, vortex to resuspend

- Incubate (at least 7’)

-  Separate on magnet 2’

- 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

QPCR Master Mix (QPCR MM, 20uL RXN)

Final QPCR Program (Opticon)

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 (google docs, Illumina Library QPCR and Multiplexing)

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

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