illumina 1000000035294 DNA Prep Microbial Colony Extraction Instructions
- June 14, 2024
- illumina
Table of Contents
**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
- Prepare the following consumables:
Item | Storage | Instructions |
---|---|---|
IPB | Room temperature | Let stand for 30 minutes to bring to room temperature. |
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
-
Using a 10µl disposable inoculation loop, pick a half loopful of colonies from the bacterial culture plate.
-
Resuspend colonies in the Power Bead tube containing glass beads and nuclease-free water.
-
Fit a Vortex-Genie 2 with a vortex adapter.
-
Vortex at speed 6 for five minutes.
-
Centrifuge at 20,000 × g for two minutes. Make sure that the glass beads are at the bottom of the Power Bead tube.
-
Transfer all supernatant (~150 µl) without the glass beads to a new 1.5 ml tube.
-
Add 30µl nuclease-free water to a new 1.5 ml tube.
-
Transfer 20µl supernatant to the 1.5 ml tube containing nuclease-free water. Pipette to mix.
-
Vortex and invert IPB several times to resuspend.
-
Add 20ul IPB to the 1.5 ml sample tube containing supernatant and water.
-
Using a pipette set to50 µl, mix ten times to thoroughly mix the beads and sample.
-
Incubate at room temperature for five minutes.
-
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. -
Remove the supernatant without disrupting the bead pellet.
-
Make sure that a bead pellet sat the bottom of the tube before discarding the supernatant.
-
If the beads are accidentally aspirated:
- a Return the sample to the tube and allow it to settle.
- b Remove and discard the supernatant.
-
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.
-
Remove any residual Ethos using a P20 pipette.
-
Air-dry the tubes on the magnetic stand for five minutes.
-
Remove the tubes from the magnetic stand.
-
Resuspend IPB in 32.5 µloft RSB. Pipette to mix.
-
Incubate at room temperature for 2 minutes.
-
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
-
Transfer 30µlto a new 1.5 ml tube.
-
Add 20µlof nuclease-free water to a 96-well PCR plate.
-
Transfer 10µlof sample to the 96-well PCR plate containing water. Pipette to mix.
-
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
Document | Date | Description of Change |
---|---|---|
Document # 10000000332941 v02 | October 2023 | Updated kit name from Nextera DNA |
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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