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Mapping with Polymorphisms

snip-SNP mapping with CB4856 polymorphisms


Materials:

Procedure:

  1. Generate recombinant lines for the mutation of interest
    • Mate your strain homozygous for the mutation of interest with the polymorphic strain CB4856
    • Pick 6-8 F1 cross-progeny to individual plates
    • Pick F2 animals homozygous for your mutation to individual plates to establish lines
      • For maternal-effect mutations, pick all F2s to individual plates and keep the ones showing the mutant phenotype
  2. Prepare template DNA for SNP-analysis
    • Pick mutant F2 animals individually into 20 ul H2O in PCR tubes
    • Add 20 ul of 2X worm lysis buffer (w/ freshly added proteinase K) to each tube
    • Process template as for Single Worm PCR
  3. Amplify polymorphic region by PCR
    • Aliqout 22.5 ul of PCR reaction mix to individual tubes
    • Add 2.5 ul of template DNA to each

      component

      final conc.

      vol per rxn

      mix for 50 rxns

      PCR Buffer

      1 X

      2.5

      137.5

      10X Buffer

      dNTPs

      100 uM / each

      0.1

      5.5

      25 mM dNTPs

      forward primer

      0.5 uM

      0.625

      34.5

      20 uM primer

      reverse primer

      0.5 uM

      0.625

      34.5

      20 uM primer

      template DNA

      (2.5)

      --

      DNA template

      Taq Polymerase

      0.5 - 1.0 U

      0.1

      5.5

      Taq

      18.55

      1021

      H20

    • Run PCR reaction for 35 cycles with the following parameters:
      • 10 secs @ 94 degrees, 10 secs @ 55 degrees, extension time (approx 30 secs/500bps) @ 72
  4. Determine polymorphism carried by each recombinant worm using restriction digests
    • Add 8 ul 2X restriction mix to 8 ul PCR product (contains 50 mM KCl, 10 mM Tris 8.3, 1.5 mM MgCl2)
      • 2X restriction mix (volumes for 50 rxns)

      ideal restriction enzyme buffer

      NEB#2

      NEB#3

      component

      stock soln

      conc in 2X

      vol of stock

      conc in 2X

      vol of stock

      NaCl

      5 M

      50 mM

      4.5 ul

      150 mM

      13.5 ul

      Tris (pH7.5)

      1 M

      10 mM

      4.5 ul

      10 mM

      4.5 ul

      MgCl2

      1 M

      18.5 mM

      8.3 ul

      18.5 mM

      8.3 ul

      DTT

      .2 M

      2 mM

      4.5 ul

      2 mM

      4.5 ul

      BSA

      100X

      200 ng/ul

      9 ul

      200 ug/ul

      9 ul

      enzyme

      1 U

      45 U

      1 U

      45 U

      H2O

      420 ul

      411 ul

    • Incubate @ appropriate temperature for 3-4 hours
    • Add 2 ul 10X loading dye to each
    • Load digests onto 2% agarose gels and run until bromophenol blue (runs < 150 bps) and xylene cyanol (runs > 1000 bps) dye fronts are well separated
    • Determine presence/absence of polymorphisms by bands observed
      • Can actually identify animals homozygous for each polymorphic type as well as heterozygotes

 

Notes & Misc:

 

References:

  1. Potential SNPs can be found at the Sequencing Center at Washington University
  2. Snip-SNPs: a rapid and powerful mapping tool. Stephen R. Wicks, Ronald H.A. Plasterk. Worm Breeder's Gazette 16(1): 28 (October 1, 1999)
  3. snip-SNPs II: Mapping using bulked segregant analysis. Stephen R. Wicks, Ronald H.A. Plasterk. Worm Breeder's Gazette 16(2): 24

 

compiled by Chad Rappleye 3/00

Aroian Lab Protocols