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Genotyping
Published: Dec 11, 2019 Views: 1164
Original research article
The authors used this protocol in:
Sep 17, 2019
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- 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
- For 100-200 bp PCR product, 7% PAGE-gel is used
- (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).
- PCR set-up
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