illumina 1000000035294 DNA Prep Microbial Colony Extraction Instructions

**illumina 1000000035294 DNA Prep Microbial Colony Extraction Instructions

**

Introduction

The following protocol demonstrates how to proceed from plated microbial cultures directly to Tag mentation as described in the Illumina DNA Prep Reference Guide (document # 10000000025416)without upstream quantitation. This protocol provides greater than 100 ng input at its conclusion.

DISCLAIMER

The information in this Illumina Demonstrated Protocol is being provided as a courtesy. In some cases, reagents are required thobe purchased from non- authorized third-party suppliers. Illumina doesn’t guarantee or promise technical support for the performance of our products used with any reagent purchased from a non-authorized third-party supplier.

Consumables

• IPB (Illumina Purification Beads) Illumina catalog # 20060057 or 20060058
• RSB (Resuspension Buffer)
• PowerBead tubes, glass (0.5 mm) (QIAGEN, catalog # 13116-50)
• Disposable inoculation loop (10 µl) (Fisher, catalog # 12870155)
• 96-well PCR plate
• Blood agar plate
• 1.5 ml tube
• 80% ethanol
• Nuclease-free water

Bacterial Colonies

The bacteria are grown overnight at 37°C on a blood agar plate. This protocol assumes sufficient colonies to fill half of10 µl loop from each bacterial culture plate.

This protocol is validated for the following bacteria:

• Pseudomonas aeruginosa
• Klebsiella pneumoniae
• Enterobacter cloacae
• Escherichia coli
• Acinetobacter baumannii
• Enterococcus faecalis
• Streptococcus agalactia
• Staphylococcus aureus

Equipment

Equipment: Supplier
Magnetic Stand-96: Thermos Fisher Scientific, catalog # AM10027
Vortex-Genie 2: Sigma, catalog # Z258423
Vortex Adapter for 1.5–2.0 ml tubes (24) : QIAGEN, catalog # 13000-V1-24

Preparation

1. Prepare the following consumables:

Keep at room temperature.
Power Bead| Room| Add 300 µl of nuclease-free water to the Power Bead tubes containing 0.5mm glass beads.
Tubes| temperature

• IPB is included in the Illumina DNA Prep kit or may be purchased separately.

Colony-based Microbial Extraction

1. Using a 10µl disposable inoculation loop, pick a half loopful of colonies from the bacterial culture plate.

2. Resuspend colonies in the Power Bead tube containing glass beads and nuclease-free water.

3. Fit a Vortex-Genie 2 with a vortex adapter.

4. Vortex at speed 6 for five minutes.

5. Centrifuge at 20,000 × g for two minutes. Make sure that the glass beads are at the bottom of the Power Bead tube.

6. Transfer all supernatant (~150 µl) without the glass beads to a new 1.5 ml tube.

7. Add 30µl nuclease-free water to a new 1.5 ml tube.

8. Transfer 20µl supernatant to the 1.5 ml tube containing nuclease-free water. Pipette to mix.

9. Vortex and invert IPB several times to resuspend.

10. Add 20ul IPB to the 1.5 ml sample tube containing supernatant and water.

11. Using a pipette set to50 µl, mix ten times to thoroughly mix the beads and sample.

12. Incubate at room temperature for five minutes.

13. Place on the magnet until the beads have fully migrated to the side of the tube (~5 minutes).
    NOTE
    The color of the bacteria can make the IPB difficult to see.

14. Remove the supernatant without disrupting the bead pellet.

15. Make sure that a bead pellet sat the bottom of the tube before discarding the supernatant.

16. If the beads are accidentally aspirated:

    • a Return the sample to the tube and allow it to settle.
    • b Remove and discard the supernatant.

17. Wash two times as follows.

    • a Add 200µl fresh 80% EtOH to each tube.
    • b Incubate on the magnetic stand for 30 seconds.
    • c Remove and discard all supernatant from each tube.

18. Remove any residual Ethos using a P20 pipette.

19. Air-dry the tubes on the magnetic stand for five minutes.

20. Remove the tubes from the magnetic stand.

21. Resuspend IPB in 32.5 µloft RSB. Pipette to mix.

22. Incubate at room temperature for 2 minutes.

23. Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

24. Transfer 30µlto a new 1.5 ml tube.

25. Add 20µlof nuclease-free water to a 96-well PCR plate.

26. Transfer 10µlof sample to the 96-well PCR plate containing water. Pipette to mix.

27. Proceed directly to the Tegument Genomic DNA procedure described in the Illumina DNA Prep Reference Guide (document # 10000000025416). Start at the step adding tag mentation master mix to the sample well.

Revision History

Flex to Illumina DNA Prep. Updated Sample Purification Beads SBP to Illumine Purification Beads IPB.
Document # 10000000332941 v01| February 2018| Update to preparation step for preparation of PowerBead Tubes.
Document # 1000000035294 v00| October 2017| Initial release

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