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Inverse PCR protocol (Maddock lab)
protocol
Protocol from Maddock lab at University of Michigan. ...
Inverse PCR
troubleshooting
I am trying to amplify a restriction fragment
from genomic DNA, where I know the sequence of half of the fragment. I am
trying to amply the other half. I ligated the fragment to form circles and
trying to amplify ... ...
...
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Last update: 17-May-2008 04:37 am
Related new papers and reviews
Identification of intron/exon boundaries in genomic DNA by inverse PCR. Curr Protoc Hum Genet. 2001 May;Chapter 6:Unit 6.4 Authors: Albertsen H, Thliveris A
This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template.This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA.
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