Efficient transfer of highly resolved small DNA fragments from polyacrylamide gels to DBM paper

Levy, Allan H.; Frei, Erich; Noll, Markus

doi:10.1016/0378-1119(80)90068-2

Cited by 16 publications

(6 citation statements)

References 12 publications

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“…The following protocol deviates from previous protocols (13)(14)(15)(16)(17)(18)(19)(20)(21). We built a 38 x 46 x 20 cm transfer device from Plexiglas egg-crate louver panels (from AIN Plastics (Mt.…”

Section: Methodsmentioning

confidence: 99%

Genomic sequencing.

Church¹,

Gilbert²

1984

Proc. Natl. Acad. Sci. U.S.A.

7,556

2,703

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Section: Methodsmentioning

confidence: 99%

Genomic sequencing.

Church¹,

Gilbert²

1984

Proc. Natl. Acad. Sci. U.S.A.

7,556

2,703

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“…The DBM paper was prepared by the method of Levy tt aL (19). 32P Labellin of DNA In order to label 5'-ends of restriction fragments, the DNA was treated with calf alkaline phosphatase and labelled with -32P-ATP and T4-polynucleotide kinase (11). DNA was labelled at the 3'-ends with a -32P-dNTP and L cQoli DNA polymerase I large fragment (Klenow) as described (20).…”

Section: Dna Mappingmentioning

confidence: 99%

Structural organization of the two main rDNA size classes ofAscaris lumbricoides

1984

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The two main rDNA size classes in the genome of Ascaris lumbricoides consist of 8.8 kb and 8.4 kb long repeating units present in a quantitative ratio of roughly 10:1. They both contain the genes coding for 18 , 5.8S and 26S ribosomal RNAs. The length heterogeneity is due to a 450 bp long spacer region localized in the longer repeating unit which begins 870 bp upstream of the 5'-end 18S gene. A few additional microheterogeneities in base sequence occur at the 5'-end of the 26S gene. The 18S, 5.8S and 26S coding regions have been mapped on both the 8.8 kb and 8.4 kb repeating units and the localization of the 5'- and 3'-ends of the 18S and 26S genes has been performed by S1 protection. No intervening sequences are present in either coding region of the two main rDNA size classes.

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“…The DNA of the core particles was purified in a polyacrylamide gel and digested with various restriction enzymes known to cleave the hsp 70 gene. The restricted DNA was separated on a 4% polyacrylamide gel, transferred to DBM-paper with high efficiency (21), and hybridized to a radioactively labeled probe consisting of the entire transcribed portion of the hsp 70 gene. If the nucleosomes are phased and the restriction site is within a core particle, the two ends of the 146 base pairs of the core particle are at defined distances from the restriction site, and hence two DNA fragments are produced that complement to 146 base pairs.…”

Section: Resultsmentioning

confidence: 99%