Dahlquist:Lactase Persistence SNP RFLP Analysis
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Reagents and Supplies
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
Standard Taq polymerase
Phusion Polymerase
FaqI Restriction Enzyme
- FaqI (isoschizomer of BsmFI): Thermofisher cat# ER1811, 200 units/μL
- 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
BsmFI Restriction Enzyme
- BsmFI (isoschizomer of FaqI): New England Biolabs cat# R0572S or R0572L, 200 units/μL
- Same recognition site as shown for FaqI above.
Overview
- Prepare cheek cell lysate according to this protocol or DNA extraction from hair follicles according to this protocol.
- Perform PCR to amplify the 448 bp region surrounding the 13,910 C>T SNP using one of the two protocols:
- Option 1: PCR using standard Taq polymerase
- Option 2: PCR using Phusion polymerase
- Run agarose gel to detect PCR products
- Purify PCR products
- Perform restriction digest using one of the two protocols:
- Option 1: FaqI
- Option 2: BsmFI
- Run agarose gel to detect restriction fragments
Option 1: PCR using standard Taq
Reactions
Number 0.2 mL PCR tubes and add template to each tube (5 μL each).
- negative control, no template, use 5 μL sterile MilliQ water in its place
- CC plasmid positive control (1:200 dilution), 5 μL
- TT plasmid positive control (1:200 dilution), 5 μL
- CT mixed plasmid positive control (1:200 dilution), 5 μL
- HeLa genomic DNA (5 ng/μL), 5 μL
- samples 6-n, cheek cell or hair follicle lysate (5 μL)
Flash spin tubes to bring templates to bottom of the tube.
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. This part can be done ahead while the template is being added to each PCR tube by another person. Do not add the Taq until that is ready and the thermocycler is ready.
- Turn on the thermocycler.
- Run "LACTASE" program (Bio-Rad S1000 thermocycler in FEA 263).
- Once lid has reached 100°C and the block has reached 94°C, pause the program by pressing the "pause" button.
- Remove enzyme cooler from freezer. Spin down Taq. Add the Taq to the master mix. Mix and flash spin.
- Add 45 μL of master mix to each PCR tube. Flick to mix and flash spin.
- Place tubes in thermocycler and un-pause program by pressing the "pause" button again.
X1 cheek cell/hair follicle lysate (5.0 μL) 10X Apex Taq Buffer 5.0 μL 50 mM MgCl2 1.5 μL 10 μM forward primer 1.0 μL 10 μM reverse primer 1.0 μL 10 mM dNTPs 1.0 μL sterile MilliQ water 35.0 μL Apex Taq DNA polymerase (5 units/μL) 0.5 μL Total 50.0 μL
Thermocycler Program
Program name on Bio-Rad S1000 thermocycler in FEA 263 is "LACTASE"
- 94°C 2 minutes
- 94°C 30 seconds
- 55°C 30 seconds
- 72°C 60 seconds
- repeat steps 2-4 for 35 cycles
- 72°C 2 minutes
- hold at 6°C
Option 2: PCR using Phusion Taq
Reactions
- Number 0.2 mL PCR tubes.
- Add 15 μL of PCR-quality water to each tube.
- Add 2.5 μL each of 10 μM Lac-13910-for-EM and Lac-13910-rev-EM primers.
- Add 5 μL template to each tube according to the list below.
- negative control, no template, use 5 μL sterile MilliQ water in its place
- CC plasmid positive control (1:200 dilution), 5 μL
- TT plasmid positive control (1:200 dilution), 5 μL
- CT mixed plasmid positive control (1:200 dilution), 5 μL
- HeLa genomic DNA (5 ng/μL), 5 μL
- samples 6-n, cheek cell or hair follicle lysate (5 μL)
- Flash spin tubes to bring templates to bottom of the tube.
Master Mix
- Turn on the thermocycler.
- Run "PHUSION" program (Bio-Rad S1000 thermocycler in FEA 263).
- Once lid has reached 100°C and the block has reached 98°C, pause the program by pressing the "pause" button.
- Remove 2X Phusion Flash High Fidelity PCR Master Mix (Cat# F548S) from freezer and thaw on ice. Mix and flash spin (gently, do not vortex!)
- Add 25 μL of the 2X Phusion Master Mix to each tube. Flick to mix and flash spin.
- Place tubes in thermocycler and un-pause program by pressing the "pause" button again.
Thermocycler Program
Program name on Bio-Rad S1000 thermocycler in FEA 263 is "PHUSION"
- 98°C 10 seconds
- 98°C 01 seconds
- 64°C 05 seconds
- 72°C 15 seconds
- repeat steps 2-4 for 35 cycles
- 72°C 1 minute
- 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 Fermentas Gene Ruler 1 kb DNA Ladder Plus (cat# SM1331) to left-most lane.
- Load the rest of the samples into the wells.
- Run gel at 120 volts until bromophenol blue has migrated 2-3 cm.
- Expect no product for the negative control and 448 base pair product for the rest of the samples.
PCR Product Purification
- Discard the negative control tube.
- Label the tubes you will need.
- The remaining 45 μL of the rest of the PCR products need to be purified before performing a restriction digest.
- Use the DNA Clean & Concentrator-5 Kit (Zymo Research, catalog D4014; Genesee Scientific, catalog 11-303C)
- In a 1.5 mL microcentrifuge tube, add 5 volumes of DNA Binding Buffer to each volume of DNA sample.
- I.e., to the 45 μL left of the PCR reaction, add 225 μL of DNA Binding Buffer.
- Mix briefly by vortexing/flash spin.
- Transfer mixture to a Zymo-Spin Column in a Collection Tube.
- Spin for 30 seconds at 10,000 X g in the microcentrifuge.
- Discard the flow-through and replace the column in the collection tube.
- Add 200 μL of DNA Wash Buffer to the column.
- Spin for 30 seconds at 10,000 X g in the microcentrifuge.
- Discard the flow-through and replace the column in the collection tube.
- Repeat the wash step by adding 200 μL of DNA Wash Buffer to the column.
- Spin for 30 seconds at 10,000 X g in the microcentrifuge.
- Discard the flow-through and replace the column in the collection tube.
- 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.
- Transfer the column to a clean 1.5 mL microcentrifuge tube with the cap cut off.
- Elute the DNA by adding 20 μL of DNA Elution Buffer directly to the column matrix
- Incubate at room temperature for 1 minute.
- Spin for 30 seconds at 10,000 X g in the microcentrifuge.
- 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.
Option 1: 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 by adding 6 μL sterile MilliQ water.
- Transfer 10 μL of purified 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, then SAM. 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.
Option 2: Restriction Digest with BsmFI
- Digest 0.4 μg of lambda DNA as a positive control. Bring 4 μL of 0.1 μg/mL lambda DNA to 10 μL by adding 6 μL sterile MilliQ water.
- Transfer 10 μL of purified PCR product to fresh 0.5 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 BsmFI (New England Biolabs cat# R0572S, isoschizomer with FaqI). Multiply the recipe by the number of tubes +0.5 to account for pipetting errors.
- Add the reagents in the order of water, then buffer. Mix and flash spin.
- Add the BsmFI. Mix and flash spin.
X1 DNA (10 μL) 10X rCutSmart Buffer 2 μL sterile MilliQ water 7 μL BsmFI enzyme (2 units/μL) 1 μL Total 20 μL
- Add 10 μL master mix to each tube, mix/flash spin.
- Incubate 60 minutes in thermocycler set at 65°C, followed by 10 minutes at 80°C to inactivate the enzyme. Program called
BSMFIon small thermocycler. - 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 entirety of the restriction digest and uncut DNA 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
