Professor Smalls Lecture 17.docx - Professor Smalls Lecture 17[Modern Methods in Biology I Sequences longer than 17 base pairs are likely to be very

Professor Smalls Lecture 17.docx - Professor Smalls Lecture...

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Professor Smalls Lecture 17 [Modern Methods in Biology I]: Sequences longer than 17 base pairs are likely to be very unique in a human genome. Some uses for cloned DNA: - Create new plasmids that combine different DNA fragments. - Add a promoter to the plasmid and make any RNA you want. - Sequence the DNA inserted in the plasmid. We use Sanger sequencing to sequence the DNA inserted in the plasmid. The idea, is that we do not know the sequence of DNA genomic fragment we inserted in the plasmid. Only the plasmid DNA sequence and the EcoR 1 sequence (GAATTC) between the genomic fragment and plasmid sequence. We heat the double stranded DNA plasmid, to denature it and separate it into single strands. We read the unknown genomic fragment sequence, by using the fact of the specificity of the DNA strand to make a primer that hybridizes to a part of the plasmid that is unique. We anneal a radioactively labeled DNA primer to the single stranded DNA. The primer must be complementary to the plasmid DNA. The primer should be 18 bases, since an 18 base sequence is what will be the most unique. The primer will hybridize to the 3’ end of the plasmid, and hybridize in the 5’ to 3’ direction.
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  • Fall '15
  • Eric Martin
  • DNA, ddNTPs, sequence of DNA

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