NAT2:Pharmacogenetic Diversity of Class 2015

Pick the sticker with your ID number, protocol, and a plastic box with a container and a lid from a TA. Open the plastic box, and label a lid with one sticker and put other sticker on your protocol copy. Note your number. Before lab, rinse mouth (do not eat or drink for 30 min)
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Close Oragene container with the lid tightly to avoid any spill; mix gently by inverting several times. Submit labeled container to TA
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NAT2:Pharmacogenetic Diversity of Class 2015

Goal:
Detection of a single nucleotide polymorphism (SNP) in NAT2 locus Evaluate potential risk for carriers of different genotypes

NAT2:Pharmacogenetic Diversity of Class 2015

LAB 1
-DNA extraction from saliva.

LAB 2
- Measurement of DNA concentration
- DNA purification for genotyping analysis

Methods:
Collection of saliva

Extraction of DNA
Quantification of DNA (fluorescence) Re-purification of DNA Genotyping assay (TaqMan technology) Analysis of genotyping data
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LAB 1: Principles of DNA

extraction:
Collect saliva (lymphocytes) Perform cell lysis and inhibit DNAhydrolyzing enzymes Precipitate DNA with ethanol and isolate by centrifugation
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LAB 1 Equipment:
Lid:

Oragene container

Reagents to perform cell lysis and inhibit DNA-hydrolyzing enzymes

Container to collect saliva ridge (~2 ml)

50oC incubator

LAB 1. Equipment:
Transfer pipette to transfer saliva (0.5ml) to a tube
0.5 ml

0.1 ml

LAB 1. Equipment:
Microcentrifuge tube labeled with your #ID

1.5 ml

Lab-1 Equipment: Ice bucket

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Lab-1. Equipment:
Microcentrifuge (13,000xg)

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Lab-1. Equipment:
Sterile swabs to remove ethanol

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Lab-1

Sample collection:
Collect saliva in the container Label two microfuge tubes with your ID number (the same # ID as the container) using a pen marker

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Lab-1

A. Sample collection:
After collecting saliva in the container tighten cap and invert container several times to mix preservative with saliva sample. Check if the membrane with the reagent is broken. Incubate in Incubator (room 428) @50oC for 1 hr

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Lab-1 B. DNA purification:

Take your container and transfer 0.5 ml into the pre-labeled microfuge tube 1 using transfer pipette (Wait for TA assistance) Add 20 l of Oragene reagent to the tube and mix by inverting several times Incubate 10 min on ice (Wait for TA assistance) Spin in a microfuge for 3 min @13,000xg Transfer supernatant to a fresh pre-labeled microfuge tube 2; discard the old tube 1 to the biohazard waste bag
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Lab-1 DNA purification continued:

Add 0.5 ml 95% ethanol with a transfer pipette to tube and mix by inverting several times Incubate for 10 min at room temperature Observe precipitation of DNA (Wait for TA assistance) Spin in a microfuge for 5 min Remove supernatant by inverting the tube and gently tapping it against a pile of paper towels
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Lab-1 DNA purification continued:

Add 0.5 ml 70% ethanol with a transfer pipette to tube with pellet and mix by inverting several times (Wait for TA assistance) Spin in a microfuge for 1 min Remove supernatant by inverting the tube and gently tapping it against a pile of paper towels. Remove traces of ethanol with a swab
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Lab-1 DNA purification continued:

Leave in inverted position for 10 min
Add 100 ul TE buffer, make sure DNA pellet is covered with buffer, and submit to TA DNA concentration will be determine at LAB 2

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