Dahlquist:Lactase Persistence SNP RFLP Analysis

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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)

GGATGCACTG CTGTGATGAG GTATCAGAGT CACTTTGATA TGATGAGAGC
AGAGATAAAC AGATTTGTTG CATGTTTTTA ATCTTTGGTA TGGGACATAC
TAGAATTCAC TGCAAATACA TTTTTATGTA ACTGTTGAAT GCTCATACGA
CCATGGAATT CTTCCCTTTA AAGAGCTTGG TAAGCATTTG AGTGTAGTTG
TTAGACGGAG ACGATCACGT CATAGTTTAT AGAGTGCATA AAGACGTAAG
TTACCATTTA ATACCTTTCA TTCAGGAAAA ATGTACTTAG ACCCTACAAT
GTACTAGTAG GCCTCTGCGC TGGCAATACA GATAAGATAA TGTAGCCCCT
GGCCTCAAAG GAACTCTCCT CCTTAGGTTG CATTTGTATA ATGTTTGATT
TTTAGATTGT TCTTTGAGCC CTGCATTCCA CGAGGATAGG TCAGTGGG

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) +2 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

First Agarose Gel

  • Pour 1.5% agarose gel with 1X TAE Buffer 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 and run gel at 120 volts.
  • Expect product of 448 base pairs for samples and ~500 bp for LOC1 positive control.

PCR Product Purification

  • The remaining 20 μL of the PCR product needs to be purified before performing a restriction digest.
  • Use the PrepEase Gel Extraction Kit (Affymetrix/USB catalog 78756), protocol for direct purification of PCR samples.
  1. Since the sample volume is < 50 μL, raise the volume to 50 μL with TE Buffer, pH 7.5, mix/flash spin.
  2. Mix 2 volumes (100 μL) of NT Buffer per 1 volume of sample. Mix/flash spin.
  3. Place a PrepEase column into a 2 mL collecting tube.
  4. Load the sample directly to the center of the clean-up column.
  5. Spin for 1 minute at 11,000 X g in the microcentrifuge.
  6. Discard the flow-through and place the clean-up column back into the collecting tube.
  7. Wash the column by adding 600 μL NT3 Buffer directly to the clean-up column.
  8. Spin for 1 minute at 11,000 X g in the microcentrifuge.
  9. Discard the flow-through and place the clean-up column back into the collecting tube.
  10. Dry column by spinning for 2 minutes at 11,000 X g in the microcentrifuge to remove excess NT3 buffer. Make sure the column does not come in contact with the flow-through when removing it from the microcentrifuge and collecting tube.
  11. Elute DNA by placing the clean-up column into a clean 1.5 mL microcentrifuge tube with the cap cut off.
  12. Add 20 μL of NE Buffer and incubate at 1 minute at room temperature
  13. Spin for 1 minute at 11,000 X g in the microcentrifuge.
  14. Check and record volume of eluate as you transfer 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

  • 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).
  • On ice, make restriction digest Master Mix with the restriction enzyme FaqI (ThermoFisher catalog FD1814, isoschizomer with BsmFI).
                               X1
DNA                          (10 μL)
10X FastDigest Green Buffer    2 μL
sterile MilliQ water           7 μL
FaqI enzyme                    1 μL
Total                         20 μL
  • Note that FastDigest Green Buffer allows direct loading of fragments on gel because it has loading dye incorporated in the buffer.
  • Add 10 μL master mix to each tube, mix/flash spin.
  • Incubate 15 minutes in 37°C water bath. Either store at -20°C or proceed directly to run on the gel (below).
  • Add 1 μL of 10X FastDigest Green Buffer to uncut DNA samples so that they can also be loaded on the gel.

Second Agarose Gel

  • Pour 1.5% agarose gel with 1X TAE Buffer and final concentration of 0.5 μg/mL ethidium bromide (50 mL total volume of gel).
  • Load entire 20 μL of restriction samples on gel. Load reserved uncut PCR products next to the appropriate sample to facilitate easy comparison.
  • 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