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the use of a gel-based approach to thoroughly remove the primer dimers.
33| Add 4 µl of 6× DNA loading buffer to the sample, mix it well by vortexing and spin down the mixture briefly.
34| Pour freshly prepared 1× TBE running buffer into the electrophoresis apparatus, and place the gel cassette into the
apparatus. Then, pull out the combs carefully to avoid destroying the wells.
35| Load 24 µl of well-mixed samples and 2 µl of low-range DNA ladder into different wells. Run the gels at 150 V for ~1 h,
stopping before the bromophenol blue loading dye runs out of the gel.
36| After electrophoresis, remove the gel cassette from the electrophoresis apparatus, peel the gel off the glass and stain it
with a 1:10,000 dilution of SYBR Gold nucleic acid gel stain in 1× TBE buffer for 10 min at room temperature in darkness on
a shaker.
nature protocols | VOL.10 NO.5 | 2015 | 653
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protocol
37| Wash the gel twice with 1× TBE buffer to remove the stain dye, and then visualize the gel with a Dark Reader
transilluminator in darkness (Fig. 2a).
38| Excise the gel slices containing 200–700-bp DNA fragments using a clean disposable blade, and then transfer the gel
slices into 0.5-ml thin-walled PCR tubes.
! caut Ion Handle the blade with appropriate protection.
39| Recover DNA fragments from the gels using an appropriate method. We have found that we achieve maximum recovery
using the ‘crush and soak’ method (Box 2).
40| Purify the eluate from Step 39 using the QIAquick PCR purification kit (or any equivalent kit) according to the
manufacturer’s protocol. Elute the DNA from the column using 50 µl of preheated EB buffer (part of the QIAquick PCR
purification kit).
41| Purify the eluate twice using 1:1-fold AMPure XP beads, as described in Box 1, and finally elute in 20 µl of
nuclease-free water.
 cr ItIcal step There will be some residual primer dimers even after the gel purification, necessitating this AMPure XP
beads purification step.
? trou Bles Hoot InG
 pause poInt The final eluate could be stored at −20 °C for no longer than 3 months.
a b
Marker #1 #2 #3 Marker 9,760
9,725 LM Single mESC RRBS UM 6,000
700 bp 9,700 3,000
2,000
9,675 1,5001,200
9,650 1,000900
800
200 bp 9,625 700
9,600 600
9,575 500
100 bp 9,550
400
9,525
9,500 300
9,475
9,450 200
9,425 100
9,400
9,378 35
Size (bp)
c
9,820
9,800 6,000
Figure 2 | The size distribution of LM Single mouse pronucleus RRBS UM
the typical scRRBS libraries. (a) The 9,750 3,000
2,000
polyacrylamide TBE gel results of 9,700 1,5001,200
1,000900
three scRRBS libraries (before 9,650 800700
gel-based size selection); the DNA is 600
smeared from 200 bp to ~5 kb, with 9,600 500
some detectable adapter dimers at 9,550
~120 bp. (b,c) The Fragment Analyzer 400
results of two scRRBS libraries 9,500 300
established from a single mESC (b) 9,450
and a single mouse pronucleus (c). 200
Typical DNA size distribution in 9,400 100
scRRBS libraries ranges from 160
to 350 bp, with visible peaks 9,344 35
corresponding to MspI fragments
for some repetitive elements. Size (bp)
654 | VOL.10 NO.5 | 2015 | nature protocols
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