Human CpG Island Clones
Human CpG island clones are grown on CircleGrow agar (Qbiogene-Alexis Ltd ) with
75µg/ml ampicillin.
As with all clones it is recommended that when the clones are
received they are restreaked out to obtain single colonies.
Positive clones can be stored long term in CircleGrow media and
ampicillin with 25% glycerol at -70°C
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Host Strain:
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Methylation tolerant E.coli strain XLI-BLUE MRF'
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Vector:
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pGEM-5Zf(-)
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Average insert size:
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760 bp
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Insert excision:
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NdeI
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Growth conditions:
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Circle grow + 75µg/ml ampicillin
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Primers:
Provided as a 10µM solution
5'-CGG CCG CCT GCA GGT CGA CCT TAA- 3' (Source BioScience UK Ltd.
no 3558)
5'-AAC GCG TTG GGA GCT CTC CCT TAA- 3' (Source BioScience UK Ltd.
no 3559)
Additional primer stocks can be obtained through Source BioScience
UK Ltd, quoting the above reference numbers.
Amplification of CpG island fragments:
We have found that the amplification of the CpG island fragments
can be achieved in a 20µl reaction volume in the following reaction
buffer :
PCR cycle has been successfully used for amplification of the CpG
island fragments. We strongly recommend that the conditions are
optimized, to allow for lab to lab variation.
Quality Control:
Our quality control work showed that the above primers with the
conditions stated amplified the CpG island fragments successfully.
Alternative primers, T7 and SP6 also carry out successful
amplification of inserts.
5' -TAA TAC GAC TCA CTA TAG GG- 3' (T7)
5' -GAT TTA GGT GAC ACT ATA G- 3' (SP6)
Database
The database can be found here (link to the attached
document)
References:
The construction of the CGI1 library is described in:
Purification of CpG islands using a methylated DNA binding
column
Cross SH, Charlton JA, Nan X and Bird AP.
Nature Genetics, 1994 6 (3), 236-244