BTN Protocol Guide-08

| Epigenetics
Bisulﬁ te DNA Conversion
Zymo Research Corp.
625 W. Katella Ave.
Suite #30
Orange, CA 92867 USA
http://www.zymoresearch.com
info@zymoresearch.com
Bisulﬁ te Conversion of DNA Directly From Blood, Tissue, and Cells
Overview
II. Bisulﬁ te Conversion
The EZ DNA Methylation-Direct
TM
Kit is a further reﬁ nement of our
popular EZ DNA Methylation
TM
and EZ DNA Methylation-Gold
TM
Kits.
1. Add 130 µl of the prepared CT Conversion Reagent to 20 µl of
The EZ DNA Methylation-Direct
TM
Kit features simple and reliable DNA
supernatant from Section I. Mix.
bisulﬁ te conversion directly from blood, tissue, and cells without the
2. Perform the following temperature steps: 98°C for 8 minutes,
prerequisite for DNA puriﬁ cation. The increased sensitivity of this kit
64°C for 3.5 hours, then hold at 4°C.
makes it possible to amplify bisulﬁ te converted DNA from as few as
3. Add 600 µl of M-Binding Buffer to a Zymo-Spin
TM
IC Column
10 cells or 50 pg DNA.
and then add the sample. Close the cap and mix by inversion.
4. Centrifuge at full speed (>10,000 x g) for 30 seconds. Discard
Speciﬁ cations
the ﬂ ow-through.
• Starting Materials:
5. Add 100 µl of M-Wash Buffer to the column and then centrifuge
30 seconds.
Cells: Compatible with cells from solid tissue, tissue culture,
6. Add 200 µl of M-Desulphonation Buffer to the column and wait
whole blood, buffy coat, biopsies, LCM (Laser-Capture Micro-
for 20 minutes.
Dissection) and FFPE samples, etc. The number of cells per
7. Spin at full speed for 30 seconds.
standard treatment can range from 10–10
5
cells. For optimal
8. Add 200 µl of M-Wash Buffer to the column, spin 30 seconds.
results, the cell number should be from 1 x 10
3
–8 x 10
4
cells.
Repeat this wash step.
9. Add 10 µl of M-Elution Buffer directly to the column matrix.
Puriﬁ ed DNA: Samples containing 50 pg - 2 µg of DNA. For optimal
Place into a 1.5 ml tube. Spin brieﬂ y to elute the DNA.
results, the amount of input DNA should be from 200 to 500 ng.
The DNA is ready for immediate analysis or can be stored at
• Conversion Efﬁ ciency: >99.5% of non-methylated C residues are
≤-20°C for later use. Recovered DNA is ideal for PCR ampliﬁ cation
converted to U; >99.5% protection of methylated cytosines.
for downstream analyses including restriction endonuclease
digestion, sequencing, microarrays, etc.
• DNA Recovery: >80%
• Sensitivity of Detection (Lower Limit): 10 cells for successful PCR
ampliﬁ cation.
Protocol (Spin Column Format)
I. Sample Digestion with Proteinase K
1. Set up Digestion: 13 µl M-Digestion Buffer (2X)

Up to 12 µl Sample (10–10
5
cells)

1 µl Proteinase K

X µl H
2
O
26 µl Total Volume
2. Incubate at 50°C for 20 minutes.
3. Mix by ﬂ icking or vortexing the tube. Spin for 5 minutes at
10,000 x g.
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 27
Zymo Protocol.indd 27 10/26/07 11:38:43 AM
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