RsaI R6371

Protocol tips

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	One microgram of DNA was digested for 6 h with 10 units of RsaI (Promega) in a 20-μl reaction volume. The restriction fragments were blunt-end ligated with phosphorylated EcoRI linkers (Promega), using T4 DNA ligase (Promega).

Protocol tips
One microgram of DNA was digested for 6 h with 10 units of RsaI (Promega) in a 20-μl reaction volume. The restriction fragments were blunt-end ligated with phosphorylated EcoRI linkers (Promega), using T4 DNA ligase (Promega).

Publication protocol

"One microgram of DNA was digested for 6 h with 10 units
of RsaI (Promega) in a 20-ml reaction volume. The restriction
fragments were blunt-end ligated with phosphorylated EcoRI
linkers (Promega), using T4 DNA ligase (Promega). The linked
DNA fragments were modified with the bisulphite modification
kit from Oncor, according to the manufacturer’s instructions.
The deaminated restriction fragments were PCR amplified with
primers directed at the deaminated sequence of the EcoRI
linkers, the remaining half of the RsaI site (59-AATTTTGTTGTTGTTGAT-39 and 59-TTAATTCCATTACTATCAAC-39).
After 40 cycles of amplification (94°C denaturation for 1 min,
55°C annealing for 1 min, and 72°C extension for 1 min) with Taq
polymerase, the PCR products were size-fractionated on a 1%
agarose gel in TAE buffer (0.04 M Tris acetate, pH 8.3/1 mM
EDTA), and a gel slab corresponding to 200–300 bp was cut out.
The PCR products were recovered from the gel by using the
QIAquick gel extraction kit (Qiagen, Chatsworth, CA), cloned
into the pGEM T-easy cloning vector (Promega), and sequenced
by using T7 and Sp6 sequencing primers and an ABI prism 373
automated sequencer (Applied Biosystems).
Two forms of sequence are revealed by using this technique:
(i) a C-depleted, T-rich sequence corresponding to the modified strand, and (ii) a G-depleted, A-rich sequence corresponding to
the complement of the modified strand. Therefore, any C in the
modified strand or G in the complement of the modified strand
represents a 5mC residue in the original sequence, and any G or
A in the modified strand represents a G or A in the original
sequence. However, any T in the modified strand is either a C or
a T in the original sequence (as C deaminates to U and sequences
as T). Hence, the occurrences of all mCpG, mCpA, and mCpmC
can be positively identified, but it is not possible to distinguish
between mCpT and mCpC in the original sequence."

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Manufacturer protocol

Download the product protocol from Promega for RsaI R6371 below.

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