Isolasi Gen α -Amilase B. aquimaris MKSC 6.2 dengan Teknik Degenerate PCR dan Inverse PCR
0.2 ml PCR tube FRM Example protocol · 2017-02-24 · Prepare priming buffer 500 µl RBF 500 µl...
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Take 4μl Lamda Control DNA (LMD) Add 2.5 μl FRM Add 3.5 μl NFW Mix gently by inversion + spin down Incubate for 1 min at 30 °C then 1 min at 75 °C Spin down briefly Before start checklist Rapid Sequencing Kit (SQK-RAD002 with EXP-LLB001) Pipettes and tips P1000, P200, P20, P10 and P2 1.5ml Eppendorf DNA LoBind tubes 0.2 ml thin-walled PCR tubes MinION SpotON Flow cell (FLO-MIN106) Nuclease-free water (NFW) NEB Blunt/TA Ligase Master Mix (MO367) Timer Microfuge Thermal cycler at 30 °C and 75 °C Add 1 µl RAD Add 0.2 µl Blunt/TA Ligase Master Mix Incubate for 5 mins at RT The library preperation is complete and ready for loading onto the MinION MASSFLOW INSTRUCTIONS 10 μl 1.2 μl 0.2 ml PCR tube Prime the Flow Cell ready for the library to be loaded when library preparation is complete Prepare priming buffer 500 µl RBF 500 µl Nuclease-free water 0.2 ml PCR tube 0.2 ml PCR tube Before start checklist MinION™ connected to computer with SpotON Flow Cell Run platform QC in parallel to library prep PSM, RBF and LLB on ice NFW at RT Platform QC completed Activator Sample Priming and loading the library RBF FRM RAD NOTES / OBSERVATIONS DNA LoBind 11.2 μl Store on ice Computer set up to run MinKNOW Desktop Agent set up Run Name set Pre-sequencing Mix (PSM) at > 4 ng/μl Prepare the MinION for sequencing protocol This step can be run in parallel with the preparation of the library from genomic DNA to Pre-sequencing Mix Assemble the MinION and MinION Flow Cell Setup MinKNOW to run the Platform QC – name the run and start the protocol script – NC_Platform_QC.py Allow the script to run to completion and the number of active pores are reported LMD Example protocol Rapid Lamda Control Experiment for the MinION TM device using SQK-RAD002 (1/2) This protocol is provided as an example. The most up to date versions are available in the Nanopore Community; a log in will provided after a starter pack has been ordered.
Transcript of 0.2 ml PCR tube FRM Example protocol · 2017-02-24 · Prepare priming buffer 500 µl RBF 500 µl...
Take 4µl Lamda Control DNA (LMD) Add 2.5 μl FRMAdd 3.5 μl NFW
Mix gently by inversion + spin downIncubate for 1 min at 30 °C then 1 min at 75 °CSpin down briefly
Before start checklistRapid Sequencing Kit (SQK-RAD002 with EXP-LLB001)Pipettes and tips P1000, P200, P20, P10 and P2 1.5ml Eppendorf DNA LoBind tubes
0.2 ml thin-walled PCR tubesMinION SpotON Flow cell (FLO-MIN106)Nuclease-free water (NFW) NEB Blunt/TA Ligase Master Mix (MO367)
Timer Microfuge Thermal cycler at 30 °C and 75 °C
Add 1 µl RAD Add 0.2 µl Blunt/TA Ligase Master Mix Incubate for 5 mins at RT The library preperation is complete and ready for loading onto the MinION
MASSFLOW INSTRUCTIONS
10 µl
1.2 µl
0.2 ml PCR tube
Prime the Flow Cell ready for the library to be loaded when library preparation is completePrepare priming buffer
500 µl RBF500 µl Nuclease-free water
0.2 ml PCR tube
0.2 ml PCR tube
Before start checklistMinION™ connected to computer with SpotON Flow CellRun platform QC in parallel to library prep
PSM, RBF and LLB on iceNFW at RTPlatform QC completed
ActivatorSample
Priming and loading the library
RBF
FRM
RAD
NOTES / OBSERVATIONS
DNA LoBind 11.2 µl
Store on ice
Computer set up to run MinKNOWDesktop Agent set upRun Name setPre-sequencing Mix (PSM) at > 4 ng/μl
Prepare the MinION for sequencing protocolThis step can be run in parallel with the preparation of the library from genomic DNA to Pre-sequencing Mix
Assemble the MinION and MinION Flow CellSetup MinKNOW to run the Platform QC – name the run and start the protocol script – NC_Platform_QC.pyAllow the script to run to completion and the number of active pores are reported
LMD
Example
protocol
Rapid Lamda Control Experiment for the MinIONTM device using SQK-RAD002 (1/2)
This protocol is provided as an example. The most up to date versions are available in the Nanopore Community; a log in will provided after a starter pack has been ordered.
FMX
RBF
FMX
Prepare the library for loading
Mix by inversion and spin down Loading the prepared library
Add 75µl of sample to the flow cell via the SpotON port in a dropwise fashion.Ensure each drop flows into the port before adding the
next.Gently replace the activator, making sure the bung enters the SpotON portClose the sample port cover and replace the MinION lid.
DNA LoBind 25.5 µl
DNA LoBind 75 µl
11 µl
Prime the Flow CellOpen the sample port. Draw back a few µls of buffer to make sure there is continuous buffer flow from the sample port across the sensor array.
Load 800 µl of the priming buffer. Wait 5 minutes
Gently lift the activator to make the SpotON port accessible Load 200 µl of the priming buffer as before
12 µl 26.5 µl
LLB
800 µl
Sample Port
200 µl
5 minutes
Starting the sequencing script in MinKNOW and the workflow in the Metrichor Agent
Return to MinKNOW, name the run, select the NC_6Hr_Lamda_Control_Exp_in_Run_FLO_ MIN106_SQK-RAD002.py (with plus_ Basecaller for local basecalling) and start in the MinKNOW dialogue boxOpen the Desktop Agent, select the latest version of the Lamda Control Experiment or SQK-RAD002, run the workflow and monitor the workflow using the visualisation options in detailsMinKNOW will report the number of pores available for sequencing before data collection begins. These may differ from those reported in the Platform QC.Allow the protocol to proceed until MinKNOW
reports Finished Successfully System Ready. Use the Stop in the Control Panel
to finish the protocol.Quit the Desktop Agent, close down MinKNOW and disconnect the MinION.
Before start checklistStore washed flow cell at 4°C or complete the returns form in the Nanopore Community
Navigate to www.metrichor.com to review the full sequencing report
Store MinION at RTReturn reagents to the freezer
MASSFLOW INSTRUCTIONS NOTES / OBSERVATIONS
25.5 µl RBF kept on ice12 µl NFW kept at RT26.5 µl LLB kept on ice11 µl Adapted and tethered library
Example
protocol
Rapid Lamda Control Experiment for the MinIONTM device using SQK-RAD002 (2/2)
This protocol is provided as an example. The most up to date versions are available in the Nanopore Community; a log in will provided after a starter pack has been ordered.
