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

Host strain:

Methylation tolerant E.coli strain XLI-BLUE MRF'

Vector:

pGEM-5Zf(-)

Average insert size:

760 bp

Insert excision:

NdeI

Growth conditions:

Circle grow + 75µg/ml ampicillin

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 :


human cpg islands 1

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 variationhuman cpg islands 2

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