Genotyping

• DNA preparation:
  • Alkaline hydrolysis: add 50 mM NaOH (50 µl, should be scaled based on the tissue size) to the sample, 95℃, 10min. Then add 1 M Tris-HCl (PH=8, 5 µl) to neutralize the sample.
  • (Alternative) Proteinase K based digestion: add 50 µl digestion buffer (50mM KCl, 2.5mM MgCl2, 10mM Tris PH=8.3, 0.45% NP40, 0.45% Tween-20, 0.01% gelatin, 0.4 mg/ml proteinase K), incubate samples at 60℃ for 2h and then inactivate Proteinase K at 95℃ for 15min.
  • Note: for many samples/primers, 1XPCR buffer can also be used with proteinase K for the digestion. DNA purification is sometimes required for specific samples/primers.

• Heteroduplex motility assay:
  • For 100-200 bp PCR product, 7% PAGE-gel is used
    • 5XTBE:                                               10 ml
    • 40% Acrylamide/Bis (19:1):                    17.5 ml
    • H₂O:                                                   71.4 ml
    • 10% APS:                                            1 ml
    • TEMED:                                              50 µl

• (Alternative) High resolution melt analysis:
  • PCR set-up
    • 5X Phusion HF buffer:                                 4 µl
    • 10 mM dNTP:                                             0.4 µl
    • 10 µM Forward/Reverse primers:                  0.4 µl/0.4 µl
    • DMSO:                                                      0.6 µl
    • SYBR-Green (100X, Invitrogen, S7563):         0.75 µl
    • DNA template:                                            1 µl
    • Add H₂O to 20 µl
  • Notes: PCR products are melted from 60 to 95°C at a rate of 0.1°C/sec. SYBR-Green dye can also be added after PCR is done in which case only melt-curve needs to be run on a real-time PCR instrument (Applied Biosystems, StepOnePlus or 7500 Fast).

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