™
Application Note Protocol 03:
Preparation of High Molecular Weight gDNA
for TrueMethyl® Processing
Version 1.1 (January 2014)
Introduction
For many DNA methylation assays, high molecular weight gDNA is required as input material for the assay. For TrueMethyl®
processing, it’s essential that the sample is presented to the TrueMethyl® workflow in a format that is compatible for successful
oxidations, free from any contaminants that the oxidant is sensitive to at specified levels. Excess ethanol from DNA extraction
methods can present a challenge, leading to adverse conditions for the oxidation reaction and suboptimal results. To eliminate
these challenges, we have developed an optimised method to prepare any high molecular weight gDNA sample for TrueMethyl®
processing without compromising the oxidation reaction.
Application Note Protocol 03: TrueMethyl® + HMW gDNA (V1.1 Jan14):
The purpose of this protocol is to provide preliminary recommended guidelines describing how to prepare a high molecular weight
sample for TrueMethyl® processing. We have used a g-TUBE (Covaris, part numbers 520079, 520104) in this version of the
Application Note Protocol, however other shearing devices could be used but have not currently been comprehensively tested by
our R&D group. Please Note: This is a major deviation from the standard TrueMethyl® workflow and has not been fully validated.
This document will be updated with improvements determined by the CEGX R&D group.
NOTE: The information provided in this document is for guidance and advice purposes only. CEGX does not guarantee success
when implementing the suggestions made in this document. We strongly recommend conducting an independent pilot study on a
standard proxy sample for reassurance purposes prior to conducting any experiments with novel deviations on precious samples.
Required materials:
g-TUBE (Covaris, part numbers 520079, 520104)
GeneJET PCR Purification kit (Thermo Scientific part numbers K0701, K0702)
Ultra pure water
50 mM NaOH
80% Acetonitrile (make an 80% solution in ultra pure water using Acetonitrile from Fisher Scientific Product Code 10010010)
Qubit dsDNA Assay Kit (Invitrogen Catalog number Q32850)
Qubit ssDNA Assay Kit (Invitrogen Catalog number Q10212)
TrueMethyl® kit (Cambridge EpiGenetix) and User Supplied components
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1. Quantitate DNA: Quantify and QC check input high molecular weight genomic DNA using the Qubit dsDNA Assay according to
the manufacturers instructions.
2. For high molecular weight genomic DNA, a pre-shearing step is necessary to enable efficient recovery and buffer exchange of
the sample from the BioRad® P6 Micro-Bio spin column used in the oxBS workflow. This protocol has been validated using a
g-TUBE (Covaris, part numbers 520079, 520104) to pre-shear 4 μg HMW gDNA to 10 kb using the manufacturers instructions.
Other shearing methods may be used and should be compatible but have not been tested for the purpose of this protocol. For
shearing efficiency it’s essential that the manufacturers recommended shearing volume (150 μl) and instructions are adhered to,
shearing with < 150 μl will not provide satisfactory results.
3. The resulting volume of sheared material is 150 μl. This volume must be reduced from 150 μl to 40 μl to make it suitable for
TrueMethyl® processing. To achieve this, a GeneJET purification column (GeneJET PCR Purification kit part number K0701,
K0702) may be used as follows:
PLEASE NOTE: this purification method has only been validated using 4 μg per GeneJET purification; any deviation from this
must be tested prior to use to ensure sufficient recovery of the 10 kb sheared DNA from the GeneJET column.
a) Pre-warm 500 μl ultra pure water to 65 °C for 30 mins using a heatblock. This will be needed for the final elution step.
b) Pre-wash the GeneJET column. One GeneJET purification column is required per 4 μg of 10kb sheared sample. Place a
GeneJET column into a collection tube and add 800 μl ultra pure water containing 50mM NaOH. Centrifuge for 60 secs at
14,000 rpm. Discard flow through.
c) Repeat step b) to complete 2 x pre-washes in total.
d) Recondition the GeneJET column by adding 800 μl of Binding Buffer (no DNA yet!) from the GeneJET kit to the column.
Centrifuge for 60 secs at 14,000 rpm. Discard flow through. The GeneJET column is now ready for use.
e) To the 150 μl of 10 kb sheared sample, add 150 μl of Binding Buffer from the GeneJET kit and mix thoroughly (i.e. use a
1 to 1 ratio of sample to Binding Buffer). Apply the entire 300 μl volume to the pre-washed and reconditioned GeneJET
column. Centrifuge for 60 secs at 14,000 rpm. Discard flow through.
f) Wash the GeneJET column by adding 800 μl of 80% Acetonitrile to the column. Centrifuge for 60 secs at 14,000 rpm.
Discard flow through. (Do NOT wash the column using an ethanol based wash solution, this will adversely affect results!)
g) Repeat step f) a further two times to complete 3 x 80% Acetonitrile washes in total.
h) Perform a "dry spin" by centrifuging the column for 60 secs at 14,000 rpm.
i) Place the GeneJET column into a new 1.5ml tube. Add 40 μl of pre-warmed ultra pure water (to 65 °C) to centre of the
GeneJET column membrane. Incubate for 2 minutes at room temperature. Centrifuge for 60 secs at 14,000 rpm.
j) To maximize yield, transfer the eluate back onto the GeneJET column. Incubate for 2 minutes at room temperature.
Centrifuge for 60 secs at 14,000 rpm. Discard the GeneJET column and retain the recovered eluted DNA sample.
k) Measure the concentration of the recovered sample using the Qubit dsDNA Assay according to the manufacturers
instructions. Record the final volume of the recovered sample. Ensure you have sufficient concentration and recovery of
sample to continue with your planned experiment.
4. Use the TrueMethyl® kit and TrueMethyl® User Guide to generate oxBS and BS converted templates, use of technical replicates
is recommended. An overview of the expected recoveries can be seen in Figure 1 below.
Figure 1. Expected Sample Recovery of TrueMethyl processed high molecular weight gDNA.
Sample gDNA
(> 20 kb)
4 μg
4 μg
Shear gDNA
(~10 kb)
(150 μl of 26.5 ng/μl)
Purify on
GeneJET column
(40 μl of 70 ng/μl)
oxBS = 1.4 μg
(20 μl of 70 ng/μl)
2.8 μg
Subdivide
sample
BS = 1.05 μg
Process through
TrueMethyl
BS = 1.05 μg
Final converted
samples
BS = 0.42 μg
(15 μl of 70 ng/μl)
Elute through
P6 column
oxBS = 1.4 μg
(23 μl of 61 ng/μl)
oxBS = 0.42 μg
(20 μl of 21 ng/μl)
(15 μl of 70 ng/μl)
(20 μl of 21 ng/μl)
Cambridge Epigenetix
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info@cegx.co.uk
technical@cegx.co.uk
© 2014 Cambridge Epigenetix Ltd
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