Ordering supplies for Shotgun DNA Mapping

Here is a list of components that are needed for this project:

  1. Anchor DNA:

    1. pRL574 – provided to us by Robert Landick
    2. pALS – purchased through DNA 2.0
    3. primers for both pRL574 and pALS
      1. F834-dig: 5′ – TTTTCCCAGTCACGACGTTG – 3′
      2. R2008-bio: 5′ – CACGTAAGGTTTCAGAGATATATGGG – 3′
      3. F50 (can order with or without Biotin): 5′ – TGTGTCGCCCTTAGGTACGAACT – 3′
      4. R4000 (can order with or without dig): 5′ – TTCGCTCCAAGCTGGGCTGTGTG – 3′
  2. Adapter DNA:
    1. Top Adapter: 5′ – GCTGTCTGAATTCTAATGTAGTATAGTAATCCGCTCATCG – 3′
      1. make sure the 5′ end is phosphorylated otherwise the ligation may fail
    2. Bottom 1a: 5′ – GAGCGGATXACTATACTACATTAGAATTCAGAC -3′
      1. X = dT-biotin
      2. Alternate versions of Bottom
        1. 5′ – TXTXTXAGAGCGGATTACTATACTACATTAGAATTCAGAC -3′
          1. X = dT-biotin
        2. 5′ – biotin – GAGCGGATTACTATACTACATTAGAATTCAGAC – 3′
  3. Molecular Biology Components
    1. PCR Supplies:
      1. Taq/OneTaq – I had amazing success with OneTaq so I may get this
      2. dNTPs
      3. MgCl2
      4. PCR buffer
    2. Digestion components
      1. BstXI
      2. SapI
      3. EarI
    3. Ligation
      1. T4 DNA Ligase
    4. Gel Components
      1. EtBr
      2. SybrSafe