Sep 16, 2024

Public workspaceT-RFLP Sephadex purification and sample preparation

DOI
dx.doi.org/10.17504/protocols.io.36wgqwyogk57/v1
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Eva Petrova
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DOI: dx.doi.org/10.17504/protocols.io.36wgqwyogk57/v1
Protocol Citation: Eva Petrova, Roey Angel 2024. T-RFLP Sephadex purification and sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqwyogk57/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2018
Last Modified: September 16, 2024
Protocol Integer ID: 12442
Abstract
Purification of PCR-product after restriction digest. Samples proceeded on the capillary sequencer.
Guidelines
The Sephadex solution is stored in a glass bottle in the fridge. Before you start the purification let the Sephadex solution come to room temperature and shake well before each usage!
Materials
MATERIALS
ReagentMultiScreen-HV Filter Plate, 0.45 µm, clear, non-sterileMerck MilliporeCatalog #MAHVN4550
ReagentSephadex G-50 M DNA Grade, 100 gGe HealthcareCatalog #17004502
Before start
Materials:
  • digested PCR-product (100-200ng in 20ul)
  • 96 well filter plates
  • 96 well collection plates (IBL 96*0.2ml frosted Subkirted thin well)
  • Sephadex G50 (GE, 37.5g into 500ml autoclaved MilliQ H2O)
  • Multipette 10ml/1ml
  • Multichanel 200ul/10ul
  • HiDi Formamide (Prepare a master mix: 10ul HiDi Formamide + 0.3ul Size Standard, GS_1200 LIZ, AB per sample nebo 165ul HiDi Formamide + 5ul Size Standard for one run = 16 samples)
Sephadex purification
Sephadex purification
  • Load 200μl Sephadex solution onto the 96 well filter plates (shake the Sephadex solution well before each usage!) (use the EppendorfMultipette with a 10ml tip), the no. of wells you load depends on the no. of samples,
  • load filter plate on 96 well collection plate, 
  • place the lit on top of this 'plate sandwich' and
  • place in swing out rotor base.
Amount200 µL Sephadex solution
Spin at 230rcf(g) for 1 min 15sec (Eppendorf centrifuge 5810). Don't forget to balance the centrifuge by placing a dummy  'plate sandwich'  on the rotor.
Command
420 x g
CENTRIFUGE
Duration00:01:00
Discard flow through.
Repeat step 2. (Shake Sephadex!)
Go togo to step #2 repeat
Place Sephadex G-50 containing 96 well filter plate on the collection plate.
Load 20μl sample in the centre of the Sephadex column. Just drop the 20ul on tj top of the gel. Avoid to destroy the Sd. gel by penetrating it  with the pipette tip.
Amount20 µL sample
Spin at 420 rcf (g) for 1 min (Eppendorf centrifuge 5810).  Note: There will be some extra buffer that comes through from the Sephadex resulting in a total sample volume of ∼ 35-40 μl.
Duration00:01:00
Command
420 x g
CENTRIFUGE
Transfer the purified samples back to the original caps.
Remove the Sephadex from the filter plate and rinse the filter plate and collection plate as well (with MQ). Both plates could be reused several times.
Go immediately to sample preparation or store the samples at -20°C.
Sample preparation for capillary sequencing
Sample preparation for capillary sequencing
Aliquot 10μl of HiDi Formamide master mix into a new 96 well plate.
Add the 4μl of your purified sample per well.
Amount4 µL purified sample
Denature samples for 3min at 95°C.
Duration00:03:00 denaturation
Temperature95 °C
Transfer samples immediately on ice and let the chill for 5 min.
Temperature0 °C on ice
Duration00:05:00
Store samples in the fridge, prepare the sample sheet and run the samples at ABI 3130XL Capillary Sequencer.