Antimicrobial Resistance Screening Design

@Amanda Dougherty

Illumina DNA Prep

• Measure DNA concentration via Synergy HTX

  • If samples are above 16 ng/ul, normalize that sample between 4 and 16 ng/ul.

    • If more than 5 samples per plate, use Nimbus to normalize to 10 ng/ul

• Add 30 ul of each sample to hard shell 96 well plate

• Bring BLT and TB1 to RT

• Add 20 ul Tagmentation Master Mix after vortexing:

• Mix via pipetting 10x

• Seal, quick spin, and run on cycler program TAG:

• Heat TSB at 37 C for 10 minutes if precipitate observed

• Spin down tagmentation plate

• Add 10 ul TSB to each well, mix by pipette 10x

• Seal, quick spin, and run on Cycler Program:

• Place plate on magnetic stand and allow to clear 3 minutes

• Remove and discard supernatant

• Remove plate from magnets

• Slowly add 100 ul TWB directly to beads, pipette slowly until resuspended

• Place plate on magnetic stand and allow to clear 3 minutes

• Remove and discard supernatant

• Remove plate from magnets

• Slowly add 100 ul TWB directly to beads, pipette slowly until resuspended

• Place plate on magnetic stand and allow to clear at least 3 minutes, leave until ready next MM is ready to add:

• Remove Supernatant

• Remove from magnet

• Add 40 ul MM from above and pipette to resuspend

• Seal and quick spin

• 10 ul pre paired i7 and i5 index adapters

• Pipette 40 ul up and down to mix

• Run on Cycler Program:

Cleanup:

• Equilibrate Sample Purification Beads to RT for 30 minutes and invert to resuspend

• Place PCR plate on magnetic stand and wait 5 minutes

• Transfer 45 ul to deepwell or midi plate

• Add 45 ul SPB to each well, pipette up and down 10x

• Incubate at RT for 5 min

• Place on magnet for 5 minutes

• Add 15 ul SPB to new midi plate

• Transfer 125 ul supernatant to new midi plate from above and mix 10x

• Discard 1st plate

• Incubate at RT for 5 minutes

• Place on magnet for 5 minutes

• Remove and discard supernatant, without moving plate

• Add 200 ul EtOH

• Incubate 30s

• Remove 200 ul

• Add 200 ul EtOH

• Incubate 30s

• Remove 200 ul

• Remove Residual EtOH

• Air Dry for 5 minutes

• Remove from magnet

• Add 32 ul RSB and pipette to resuspend

• Incubate at RT 2 minutes

• Return to magnet 2 minutes, Transfer 30 ul to new pcr plate.

Spot Check preps via qPCR and/or bioanalyzer

MyBaits:

1st Hybridization

• Pool 4+ libraries together in equal volumes and concentrate with SpeedVac leaving at least 7 ul

• Prepare Hybridization MM:

• Incubate Hyb MM at 60C for 10 min

• Incubate at RT for 5 min

• Add 18.5 ul Hyb MM to Hyb rxn tubes, HYB (0.2 ml)

• Add 5 ul Blockers Mix to separate LIB tube:

• Add 7 ul of a library pool to each Blocker Mix (LIB) and mix by pipette

• Add LIBs to Cycler running:

• Once Cycler reaches temp for step 2, add HYBs to cycler

• After step 2 completes, transfer 18 ul from HYBs to each LIB

• Incubate overnight 16 - 24 hours

Wash

• Make and Heat Wash Buffer X at 60 C for >30 minutes:

1. Aliquot 30 ul beads per rxn to 0.2 ul strip tubes

2. Place on magnet for 1-2 minutes

3. Discard supernatant

4. Add 200 ul Binding Buffer, vortex to resuspend, and quick spin

5. Place on magnet for 1-2 minutes

6. Discard Supernatant and return to step 4 twice more

7. Resuspend in 70 ul Binding Buffer

8. Heat Bead Aliquots to 60C for 2 minutes

9. Transfer 30 ul LIBs to Heated Beads, pipette to mix

10. Incubate for 5 minutes, agitate after 2.5 minutes

11. Transfer Tubes to magnet and allow to clear

12. Add 180 ul warmed Wash Buffer X, incubate at 60 C for 15 s, vortex and spin

13. Incubate for 5 minutes at 60 C, agitate at 2.5

14. Place on Magnet until solution clears

15. Discard supernatant

16. Return to step 12 for 4 total washes

17. Discard residual supernatant

18. Add 30 ul Buffer E And thoroughly resuspend off magnet

19. Incubate at 95 C for 5 minutes

20. Place on Magnet until solution clears and transfer 15 ul of supernatant to 2 tubes with PCR MM:

• Run on Cycler Program:

•  

2nd Hybridization

• Combine both PCRs from above and concentrate to ~7 ul

• Prepare Hybridization MM:

• Incubate Hyb MM at 60C for 10 min

• Incubate at RT for 5 min

• Add 18.5 ul Hyb MM to Hyb rxn tubes, HYB (0.2 ml)

• Add 5 ul Blockers Mix to separate LIB tube:

• Add 7 ul of a PCR pool to each Blocker Mix (LIB) and mix by pipette

• Add LIBs to Cycler running:

• Once Cycler reaches temp for step 2, add HYBs to cycler

• After step 2 completes, transfer 18 ul from HYBs to each LIB

• Incubate overnight 16 - 24 hours

Wash

• Make and Heat Wash Buffer X at 60 C for >30 minutes:

1. Aliquot 30 ul beads per rxn to 0.2 ul strip tubes

2. Place on magnet for 1-2 minutes

3. Discard supernatant

4. Add 200 ul Binding Buffer, vortex to resuspend, and quick spin

5. Place on magnet for 1-2 minutes

6. Discard Supernatant and return to step 4 twice more

7. Resuspend in 70 ul Binding Buffer

8. Heat Bead Aliquots to 60C for 2 minutes

9. Transfer 30 ul LIBs to Heated Beads, pipette to mix

10. Incubate for 5 minutes, agitate after 2.5 minutes

11. Transfer Tubes to magnet and allow to clear

12. Add 180 ul warmed Wash Buffer X, incubate at 60 C for 15 s, vortex and spin

13. Incubate for 5 minutes at 60 C, agitate at 2.5

14. Place on Magnet until solution clears

15. Discard supernatant

16. Return to step 12 for 4 total washes

17. Discard residual supernatant

18. Add 30 ul Buffer E And thoroughly resuspend off magnet

19. Incubate at 95 C for 5 minutes

20. Place on Magnet until solution clears and transfer 15 ul of supernatant to 1 tubes with PCR MM and 1 without:

• Run on Cycler Program: