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Methylated CpG island Amplification
Protocol written by Minoru Toyota
2. Materials
2.1. MCA
Restriction enzymes SmaI, XmaI
T4 DNA ligase
Taq DNA polymerase
10X PCR reaction buffer:
670mM Tris-HCl, pH 8.8
40mM MgCl2
160 mM NH4(SO4)2
100 mM b -Mercaptoethanol
1 mg/ml bovine serum albumin.
Tris-EDTA (TE) pH 8.0
DNA precipitation reagents:
Phenol/Chloroform pH 8-9
3M NaOAc (for general precipitation)
5M NH4Oac (for precipitation and quantitation when dNTPs are present)
100% ETOH
Agarose gel electrophoresis reagents
Filter hybridization reagents:
96 pin replicator system (Nunc)
Nylon membranes
DNA hybridization solution (e.g. BLOTTO)
Random-primed DNA labeling kit
Wash solutions (Wash1 2xSSC, 0.1%SDS; Wash2 0.1XSSC, 0.1%SDS)
2.2. RDA and cloning PCR products.
3 X EE buffer : 30 mM EPPS (SIGMA) pH 8.0, 3 mM EDTA pH 8.0.
5 M NaCl
cDNA spun column (Amersham)
Mung bean nuclease (NEB)
pBluescript (Stratagene)
2.3 Oligonucleotides.
RXMA primers
RXMA24 : 5’-AGCACTCTCCAGCCTCTCACCGAC-3’
RXMA12 : 5’-CCGGGTCGGTGA-3’
JXMA24 : 5’-ACCGACGTCGACTATCCATGAACC-3’
JXMA12 : 5’-CCGGGGTTCATG-3’
NXMA24 : 5’-AGGCAACTGTGCTATCCGAGTGAC-3’
NXMA12 : 5’-CCGGGTCACTCG-3’
RMCA primers
RMCA24 : 5’-CCACCGCCATCCGAGCCTTTCTGC-3’
RMCA12 : 5’-CCGGGCAGAAAG-3’
JMCA24 : 5’-GTGAGGGTCGGATCTGGCTGGCTC-3’
JMCA12 : 5’-CCGGGAGCCAGC-3’
NMCA24 : 5’-GTTAGCGGACACAGGGCGGGTCAC-3’
NMCA12 : 5’-CCGGGTGACCCG-3’
3. Methods
3.1. Preparation of MCA amplicons
3.1.1 Digestion of genomic DNA
- Digest 5 m g of genomic DNA using 100 units of SmaI over night.
- Add 20 units of XmaI and incubate at 37 ° C for 6 hours.
- Add one volume PC9, vortex, spin and extract the supernatant.
- Precipitate the DNA: Add 1/10th volume 3M NaOAc and 2 volumes 100% ETOH. Store at
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