Week 9.2
5/28/09
Molecular Epidemiology Lab (4)
Lab (3) Review:
DNA Extraction from Buccal Cell Step 3 – Purification
(1) Extract DNA Part II – Purification
(2) Dissolve DNA with storage buffer
DNA Extraction from Saliva Step 2 – Extraction and Purification
(1) Extract and purify DNA with Oragene Purifier kit
(2) Dissolve DNA with storage buffer
General overview for Lab (4):
(1) Measuring DNA concentration and the quality of DNA
(2) Set up for PCR GST M1, T1, beta-globin
(3) PCR Amplification
Time Frame of Lab (4):
Time
Event
Note
10:00 ~ 10:05
Introduction
Introduction what we will do today
10:05 ~ 10:15
Introduction
What is Spectrophotometer, how to calculate DNA
concentration and how to measure the quality of
DNA using Spectrophotometer
10:15 ~ 10:30
Dilute DNA
10 l of DNA + 90 l of sterile water
10:30 ~ 10:50
Using
Measure DNA concentration and the quality of
Spectrophotometer
DNA
10:50 ~ 11:00
Break
11:00 ~ 11:20
Introduction
11:20 ~ 11:40
PCR mix prepare
11:40 ~ 11:50
PCR set up
What is PCR
Week 9.2
5/28/09
 Instruction of Measuring DNA Concentration and the quality of DNA using
Spectrophotometer
1. Dilute 10 l of DNA with 90 l of sterile water in a 500 l microcentrifuge tube
2. Pipet this solution into the cuvette
3. Obtain measurements from spectrophotometer
Measuring DNA Concentration
a. obtain the spectrophotometer measurement at 260nm
b. calculate:
DNA concentration in μg/ml = 50 μg/ml x OD260 x 10
1000 μl /ml
10 is the dilution factor
1 OD = 50 μg/ml for double stranded DNA
1 OD = 40 μg/ml for single stranded DNA
Measuring the quality of DNA
a. obtain the ratio of OD260/ OD280
b. if the ratio > 1.8 then the DNA quality is good
if the ratio < 1.8 then the DNA quality is poor
Week 9.2
5/28/09
 Instruction of PCR assay of GSTM1/T1
1. in a sterile 0.2ml microfuge tube, mix in the following order:
H2O
8.2 l
10x buffer
2.0 l
dNTPS
1.0 l
DMSO
1.0 l
GSTT1 F
1.0 l
GSTT1 B
1.0 l
GSTM1 F
1.0 l
GSTM1 B
1.0 l
-globin F
1.0 l
-globin B
1.0 l
DNA
1.5 l
Taq DNA polymerase
0.3 l
overall
20 l
2. Carryout amplification as described.
Cycle
Denaturation
Annealing
Extension
First cycle
Subsequent cycle
(35 cycle)
5min at 94C
1min at 94C
1min at 62C
1min at 72C
Last cycle
5min at 72C
Storage 4C
**Many researchers used a 2-5 minutes first denaturing step before the actual cycling starts. This is
supposed to help denature the targeted DNA better.
**An annealing time of 30-60 seconds was sufficient for all primer pairs tested so far.
** The additional 5 minutes of extension time can help finish the elongation of most PCR products
initiated during the last cycle