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Dahlquist:Lactase Persistence SNP RFLP Analysis

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Contents

Reagents and Supplies

PCR

Primers from Morales et al. (2011) paper

Lac-13910-for-EM (below)

5'-GGA TGC ACT GCT GTG ATG AG-3'

Lac-13910-rev-EM (below)

5'-CCC ACT GAC CTA TCC TCG TG-3'
  • PCR product from primer pair "LAC 13910 EM" forward and reverse is expected to be 448 bp long.

PCR product 5' to 3' (below; primer binding sites are bolded, FaqI restriction enzyme recognition site is underlined, ^ marks the cut site, it has a four nucleotide 5' extension)

GGATGCACTG  CTGTGATGAG GTATCAGAGT CACTTTGATA TGATGAGAGC
AGAGATAAAC  AGATTTGTTG CATGTTTTTA ATCTTTGGTA TGGGACATAC
TAGAAT^TCAC TGCAAATACA TTTTTATGTA ACTGTTGAAT GCTCATACGA
CCATGGAATT  CTTCCCTTTA AAGAGCTTGG TAAGCATTTG AGTGTAGTTG
TTAGACGGAG  ACGATCACGT CATAGTTTAT AGAGTGCATA AAGACGTAAG
TTACCATTTA  ATACCTTTCA TTCAGGAAAA ATGTACTTAG ACCCTACAAT
GTACTAGTAG  GCCTCTGCGC TGGCAATACA GATA^AGATAA TGTAG C/T CCCT
GGCCTCAAAG  GAACTCTCCT CCTTAGGTTG CATTTGTATA ATGTTTGATT
TTTAGATTGT  TCTTTGAGCC CTGCATTCCA CGAGGATAGG TCAGTGGG
  • FaqI recognition site is:
5'-GGGACN10^-3'
3'-CCCTGN14^-5'
  • 5' end of forward primer to first cut site: 106 bp
  • First cut site to second cut site: 229 bp
  • Second cut site to 3' end of reverse primer: 113 bp
  • First cut site to 3' end of reverse primer: 342 bp

Reactions

Number 0.2 mL PCR tubes and add template (and primers for positive control) to each tube.

  1. negative control, no template, use 2.5 μL water in its place
  2. positive control, pRL134 plasmid template (1 μL), LOC1 forward and reverse primers (0.5 μL each)
  3. samples 3-n, cheek cell template (2.5 μL)

Master Mix

  • Make master mix on ice. Multiply the recipe by the number of tubes (listed above) +1 to account for pipetting errors.
  • Add the reagents in the order of water, buffer, MgCl2, primers, and dNTPs. Mix and flash spin.
  • Add the Taq. Mix and flash spin.
  • Add 22.5 μL of master mix to each PCR tube. Mix and flash spin.
  • Place tubes in thermocycler and start program (see below).
                                         X1
cheek cell DNA                          (2.5  μL)
10X Apex Taq Buffer                      2.5  μL
50 mM MgCl2                              0.75 μL
10 μM forward primer                     0.5  μL
10 μM reverse primer                     0.5  μL
10 mM dNTPs                              0.5  μL
sterile MilliQ water                    17.25 μL
Apex Taq DNA polymerase (5 units/μL)     0.5  μL
Total                                   25    μL

Thermocycler Program

Program name on Bio-Rad S1000 thermocycler in LSB 263 is "LACTOSE"

  1. 94°C 2 minutes
  2. 94°C 30 seconds
  3. 55°C 30 seconds
  4. 72°C 60 seconds
  5. repeat steps 2-4 for 35 cycles
  6. 72°C 2 minutes
  7. hold at 6°C

Agarose Gel to Detect PCR Products

  • Pour 1% agarose gel with 1X TAE Buffer (50 mL for small gel box/100 mL for large gel box) and final concentration of 0.5 μg/mL ethidium bromide (50 mL total volume of gel).
  • Remove 5 μL of PCR product from PCR tube and place into a 1.5 mL microcentrifuge tube. Add 1 μL of 6X loading dye. Mix and flash spin.
  • Load 10 μL of Quick-Load Purple 100 bp DNA Ladder (NEB cat# N0551S) to left-most lane.
  • Load the rest of the samples.
  • Run gel at 120 volts until bromophenol blue has migrated 2-3 cm.
  • Expect product of 448 base pairs for samples and ~500 bp for LOC1 positive control.

PCR Product Purification

  1. The remaining 20 μL of the PCR product needs to be purified before performing a restriction digest.
  2. Use the DNA Clean & Concentrator-5 Kit (Zymo Research, catalog D4014; Genesee Scientific, catalog 11-303C)
  3. In a 1.5 mL microcentrifuge tube, add 5 volumes of DNA Binding Buffer to each volume of DNA sample.
    • I.e., to the 20 μL left of the PCR reaction, add 100 μL of DNA Binding Buffer.
    • Mix briefly by vortexing/flash spin.
  4. Transfer mixture to a Zymo-Spin Column in a Collection Tube.
  5. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
  6. Discard the flow-through and replace the column in the collection tube.
  7. Add 200 μL of DNA Wash Buffer to the column.
  8. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
  9. Discard the flow-through and replace the column in the collection tube.
  10. Repeat the wash step by adding 200 μL of DNA Wash Buffer to the column.
  11. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
  12. Discard the flow-through and replace the column in the collection tube.
  13. Spin the column again at 10,000 X g in the microcentrifuge to remove excess wash buffer. Make sure the column does not come in contact with the flow-through when removing it from the microcentrifuge and collecting tube.
  14. Transfer the column to a clean 1.5 mL microcentrifuge tube with the cap cut off.
  15. Elute the DNA by adding 20 μL of DNA Elution Buffer directly to the column matrix
  16. Incubate at room temperature for 1 minute.
  17. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
  18. Check and record volume of eluate as you transfer it to a 1.5 mL tube with lid. Store at -20°C or on ice if going on to the next step immediately.


Restriction Digest with FaqI

  • Digest 0.4 μg of lambda DNA as a positive control. Bring 4 μL of 0.1 μg/mL lambda DNA to 10 μL with sterile MilliQ water.
  • Transfer 10 μL of PCR product to fresh 1.5 mL tube. (The remaining ~10 μL will be reserved as an uncut sample to run on the gel as a comparison).
  • Make restriction digest master mix on ice with the restriction enzyme FaqI (ThermoFisher catalog ER1811, isoschizomer with BsmFI). Multiply the recipe by the number of tubes +0.5 to account for pipetting errors.
  • Add the reagents in the order of water, buffer. Mix and flash spin.
  • Add the FaqI. Mix and flash spin.
                               X1
DNA                          (10 μL)
10X Buffer Tango               2 μL
50X SAM                      0.4 μL
sterile MilliQ water         6.6 μL
FaqI enzyme                    1 μL
Total                         20 μL
  • Add 10 μL master mix to each tube, mix/flash spin.
  • Incubate 60 minutes in 37°C water bath. Either store at -20°C or proceed directly to run on the gel (below).
    • Add 4 μL of 6X loading dye to the digested samples.
    • Add 2 μL of 6X loading dye to uncut DNA samples so that they can also be loaded on the gel.

Agarose Gel to Detect Restriction Fragments

  • Pour 1.5% agarose gel with 1X TAE Buffer and final concentration of 0.5 μg/mL ethidium bromide (100 mL total volume of gel for large gel box).
  • Load 10 μL of Quick-Load Purple 100 bp DNA Ladder (NEB cat# N0551S) to left-most lane.
  • Load entire 20 μL of restriction samples on gel. Load reserved uncut PCR products to the left of the appropriate cut sample to facilitate easy comparison.
  • Load the lambda DNA control digest in the right-most lane.
  • Run gel at 120 volts.
    • CC genotype: two fragments of 351 and 97 bp
    • TT genotype: three fragments of 253, 98/97 bp
    • CT genotype: four fragments of 351, 253, and 98/97 bp

Useful Links

References