MinIon testing 16 June 2014 Bacterial DNA samples

Streptomyces avermitilis DNA 75.6 ng µl-1 13.2 µl + 76.8 µl EB

Borrelia burgdorferi DNA 130 ng µl-1 7.7 µl + 82.3 µl EB

Escherichia coli DNA K-12 DNA (10798) 23ng µl-1 10 µl + 80 µl EB
1 ug in 90 µl fragmented using Covaris g-tube centrifuged at 5000 for 60s in Eppendorf 5424R
centrifuge.(AIM = 20kb)
CS DNA control added to each sample before end repair and A-tailing:
Volume CS DNA (µl)
Streptomyces avermitilis DNA
1000 ng
5
Escherichia coli DNA K-12 DNA (10798) 230 ng
1.15
Borrelia burgdorferi DNA
1000 ng
5
End repair and adenylation of fragments
Reagents used are from NEXTflex Rapid DNAseq kit (#5144-02)
Blunt end repair and adenylate the fragments by adding the following reagents to the sample in order:
Recovered DNA
96.0 µl
Nextflex End repair and adenylation buffer mix
45.0 µl
Nextflex End repair and adenylation enzyme mix
9.0 µl
150 µl
Mix by pipetting up and down carefully.
Incubate at 22°C for 20 min,
72°C for 20 min,
(4°C soak until ready)
Purification using MgNA beads and eluted with 53 µl 1x dA-tailing buffer.
1 µl was checked on a Bioanalyser 7500 DNA chip
Concentration was measured using the Qubit BR assay:
Sample
ng µl-1
Streptomyces avermitilis DNA
12
Escherichia coli DNA K-12 DNA (10798) <0.1
Borrelia burgdorferi DNA
11.05
amount (ng)
576
530
A-tailed DNA stored overnight at -20°.
N.B. E.coli DNA isolation, fragmentation and polishing required before pooling/library preparation.
Tuesday 17th June 2014
DNA was isolated from Escherichia coli DNA K-12 (10798) using the Sigma bacterial gemonic DNA
isolation kit.
1000ng was diluted in 90 µ EB and fragment by centrifuging through a g-Tube at 5000 for 60s in
Eppendorf 5424R centrifuge(AIM = 20kb).
End repair and adenylation of fragments
Reagents used are from NEXTflex Rapid DNAseq kit (#5144-02)
Blunt end repair and adenylate the fragments by adding the following reagents to the sample in
order:
Recovered DNA
96.0 µl
Nextflex End repair and adenylation buffer mix
45.0 µl
Nextflex End repair and adenylation enzyme mix
9.0 µl
150 µl
Mix by pipetting up and down carefully.
Incubate at 22°C for 20 min,
72°C for 20 min,
(4°C soak until ready)
Purification using MgNA beads and eluted with 27 µl 1x dA-tailing buffer.
1 µl was checked on the bioanalyser 7500 chip.
Concentration was measured using the Qubit BR assay:
Sample
ng µl-1
Streptomyces avermitilis DNA
12
Escherichia coli DNA K-12 DNA (10798) 20
Borrelia burgdorferi DNA
11.05
amount (ng)
576
560
530
Ligation of Oxford Nanopore adapters
dA-tailed Streptomyces avermitilis DNA
dA-tailed Escherichia coli DNA K-12 DNA (10798)
dA-tailed Borrelia burgdorferi DNA
ng µl-1 volume (µl-1)
12
18.0
20
12.0
11.05
20.0
Adapter mix
HP adapter
5x ligation buffer
T4 DNA ligase, 2000U/μl
Total
Mixed by inversion. Incubated at room temperature for 10 minutes.
10
10
20
10
100 μl
Purify the adapter ligated DNA with 0.4X Agencourt Ampure XP beads by volume. Mix thoroughly by
flicking and inversion at least 5-10 times.
3. Incubate for 15 minutes at room temperature.
4. Put the tubes on an appropriate magnetic stand to separate beads from supernatant. After the
solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to
disturb the beads that contain the DNA targets.
5. Add 150 μl of Oxford Nanopore-supplied wash buffer to the tube while in the magnetic stand.
Incubate at room temperature for 30 seconds, carefully remove and discard the supernatant.
6. Centrifuge briefly at low speed and remove any residual wash buffer
7. Air dry beads for 1-2 minutes while the tube is on the magnetic stand with the lid open.
8. Elute DNA target by adding 25.5 μl Oxford Nanopore supplied elution buffer to the beads. Mix well
by pipetting up and down very slowly, leave at room temperature for 5 min; put the tube plate in the
magnetic stand until the solution is clear.
9. Without disturbing the bead pellet, carefully transfer 25 μl of the supernatant to a fresh, sterile
microfuge tube.
Tether Annealing
25 μl
Tether
10 μl
Incubated at RT 10 min
Ligated DNA
Library Conditioning
Adapted and tethered DNA
35 μl
HP motor
15 μl
Incubated 30 min RT
This is the pre-sequencing mix, store in the fridge long term.
Immediately before running the library:
6 μl prepared library
140 μl EP
4 μl fuel mix,
Mix by inversion, centrifuge gently, before loading tonto the MinIon flowcell
Starting the MinKNOW run
Loaded the flowcell onto the device: Flowcell ID is MN-20-46087
Start software, name run (140617_bacteria_1) and MAP_Platform_QC.py configuration undertaken.
350 channels available.
Flowcell was conditioned using 2 x150 μl EP buffer with 10 min incubation each time
150 μl library was loaded and run for 48h programme, starting at 19:48
NB. After 14421sec there is a 15s pause during which new library can be added to the flowcell.
Wednesday 18th June 2014
Added more library at 12:21 during the 14421sec pause without any unforeseen consequences.