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DIAGENODE
BIORUPTOR
®
PLUS
USER MANUAL
Diagenode Inc. North America
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Troubleshooting
Bioruptor: Chromatin Shearing FAQs
Critical Steps
Questions
Answers
Comments
Fixation
What is the formaldehyde
final concentration
1%
Correct formaldehyde concentration
in fixation is critical.
How long is the fixation
step?
Fix for 10 minutes (with a time
course when needed)
It is possible to fix for as little as 5
minutes (depending on your protein of
interest for subsequent ChIP assays).
What is the temperature to
use for fixation?
Fix at room temperature
Fixation can be performed at 4°C, RT,
and 37°C. Make sure you perform the
fixation step at the right temperature.
Are the washes after
fixation important?
Wash the fixed cells properly.
Make sure you get rid of ALL the
formaldehyde. Use glycine to stop
the fixation.
Cell lysis
How can I achieve
complete cell disruption?
Do not use too many cells in the
cell lysis buffer. Lyse about 5x 10e6
cells/1 ml
The HighCell # ChIP kit is compatible
with cell numbers up to 10 million
cells in small volumes.
Number of cells/
shearing buffer
volume
What is the amount of
cells per shearing trial to
use?
1x 10e6–10x 10e6cells/ 300 μl
3x 106–30x 106 cells/ 1 ml
Do not use a too high cell
concentration.
Shearing buffer
What is the key buffer
component?
Include detergent in buffer
Quality and quantity of detergent is
important.
Shearing step
How long is the shearing?
Perform a time course for chromatin
shearing
It is possible to shear from 5-30
minutes. If 30’, interrupt sonication
after every 10 minutes and centrifuge
tubes briefly before proceeding with
the remaining time.
What is the optimal cycle?
30 seconds “ON” + 30 seconds “OFF”
What is the best
temperature for shearing?
4°C
Make sure sonication bath is kept
cool. Once optimal conditions are
reached, use for all assays to assure
reproducibility.
What is the best volume/
tube for shearing?
1.5 ml per 15 ml tube
200 μl per 1.5 ml tube
Do not use a too big sample volume
Checking for
high-quality shearing
on an agarose gel
What kind of gel should
I use to determine size
accuracy?
Check disrupted material on a 1%
agarose gel (10 μl/lane). Run the gel
slowly
Reverse cross-link from DNA after
phenol/chloroform extraction before
loading on gel.
What do smears indicate?
Gel electrophoresis of cross-linked
samples often gives smears on gel.
Also take several pictures of the gel
to assure image quality.
To obtain clearer image with accurate
fragment size, reversion of the
cross-linking is advised.
How much DNA should
I load and is RNAse
treatment necessary?
The migration of large quantities of
DNA on agarose gel can lead to poor
quality pictures which do not reflect
the real DNA fragmentation.
Do not load too much on a gel. Do not
load more than 5 μg/lane.
Also treat the sample with RNAse.
What should my running
buffer concentration be?
1X TAE or TBE is preferred to 0.5X
TAE which can lead to smears on gel.
Will using an old gel cause
problems?
Use a freshly prepared gel and fresh
buffer.
Do not reuse an old gel.
