Transcript Welcome Bio21 Online Hybrid Section 4058 Fall 2012 PLEASE KEEP THE LAB CLEAN NO FOOD/DRINKING/GUM IN THE LAB TURN OFF CELL PHONE (except when used as a.

Welcome Bio21 Online Hybrid
Section 4058
Fall 2012
PLEASE KEEP THE LAB CLEAN
NO FOOD/DRINKING/GUM IN THE
LAB
TURN OFF CELL PHONE (except when
used as a calculator)
Thomas (Tom) Chen
Ph.D. from the University of Florida
Postdoc: Colorado State University
Fellow: National Institutes of Health
Professor: The University of Tennessee, Knoxville
Visiting Professor: Medical Univ. of S. Carolina
Stanford University
My curriculum vitae is posted in Doc Sharing.
Course Home
Course Home
Syllabus
Letter of Recommendation
Threaded Discussio
Libr Assgn 1
Assessment
Lectures
Tech Info
McGraw-Hill Connect Biology
Library Assignment II
Threaded Discussion
Threaded Discussion Grading
General Comments/Questions
Test 1
Test 2*
Test 3*
Test 4*
Test 5*
Test 6*
Final Exam*
Career Choice/Advice
Lab Quiz #1
Lab quiz 2*
Library Assignment I
Library Assignment 2*
Standard Curve Lab
Diffusion Lab Report*
Enzyme Lab Protocol*
Enzyme Lab*
General Comments/Questions II*
Enzyme Lab*
Libr 2*
Libr II Due 1014*
Today
•
•
•
•
•
Class roll.
Add/Drop
Syllabus & Course eCompanion website.
Assignment Week 1
Standard Curve DNA Lab procedures
The goals of this exercise are to:
• Separate the cellular components of a plant cell to
obtain plant DNA.
• Develop a consistent process for measuring the
concentration of a component in solution.
• Process data to make a standard curve graph.
• Use the standard curve graph to determine the
concentration of the unknown DNA solution.
• Use collective standard curve data to evaluate the
variability that occurs during experimentation.
• Write a laboratory report that presents the data and
your interpretation of the data.
GOOD LABORATORY PRACTICE
• Please read and be familiar with Lab Safety Rules.
• Please handle the cuvettes and other lab instruments
with care, e.g., do not slam close the sample lid.
• Clean up everything that you used on your bench and
more importantly, the common area that you have
used.
• Do not forget to take the cuvette out of the spec when
done.
• Dispose of wastes in proper containers.
• Be careful with every step that you carried out.
• Record keeping: Record every observation that you have
made.
The exercise is divided into three parts
A. Strawberry DNA Extraction
B. Standard Curve Preparation
C. DNA Quantitation
• DNA is enclosed in a nuclear and a cell
membrane made of phospholipids
• DNA is also coiled around proteins
• Both the phospholipid layer and the
proteins must be removed to see DNA
To Extract DNA, You Must Remove …
•
•
•
•
Cell membrane
Cytoplasm
Nuclear membrane
Proteins
• strawberry in a zip-loc bag.
• 10 ml of DNA Extraction Buffer.
• plastic funnel with cheesecloth. Place the funnel
in a 150 ml beaker.
• plastic transfer pipette.
• ice-cold ethanol.
• Add 0.5 ml of 70% ethanol to a 1.5 ml microfuge
tube. 2x
• wooden stick.
• 5 drops of 0.2% methylene blue.
• tap water. 2x, 5 ml tap water.
A. Strawberry DNA Extraction
Set up the cheesecloth funnel first.
Place one strawberry in a zip-loc bag and seal the top of
the bag completely.
Mash or squash the strawberry with your hands for about
2 minutes.
A. Strawberry DNA Extraction
Add 10 ml of DNA
Extraction Buffer.
This buffer contains
detergent and salt (sodium
chloride).
Reseal and mash for 2 more
minutes.
Line a plastic funnel with cheesecloth.
Place the funnel in a 150 ml beaker.
Cut a corner from the zip-loc bag and
empty the strawberry solution into the
cheesecloth.
Gently squeeze the solution into the
funnel and allow it to collect in the
beaker. Minimize foaming
A. Strawberry DNA Extraction
Transfer one-third to one-half of the
strawberry solution to a 15 ml tube.
Using a plastic transfer pipette, slowly add
ice-cold ethanol down the side of the tube
so that it forms a layer on top of the
strawberry solution.
DNA dissolves in water, but NOT in alcohol
Continue adding the ethanol until the tube
is about three-quarters filled and you see
the DNA appearing as a white, wispy
material at the interface between the
strawberry solution and ethanol. Remove
the DNA from the tube, being careful not to
disturb the interface
A. Strawberry DNA Extraction
Add 0.5 ml of 70% ethanol to a
1.5 ml microcentrifuge tube.
Place a wooden stick into the
tube at the solution-ethanol
interface and rotate gently to
wind the DNA around the stick.
Transfer the DNA from the stick into the
70% ethanol solution. The material will
be sticky and you may need to scrape
the stick against the edge of the tube or
use the transfer pipette to push the DNA
from the stick. Use an additional 0.5 ml
of 70% ethanol to completely transfer
the DNA, ending with a total volume of 1
ml in the microcentrifuge tube.
A. Strawberry DNA Extraction
Spin the tube with the DNA
DNA forms a pellet on the side of
the microcentrifuge tube
Pour off the supernatant which is
alcohol
Let the tube air dry for 5 minutes.
Add 5 drops of 0.2% methylene
blue to the DNA pellet. Use the
wooden stick or a transfer
pipette to mix the dye with the
DNA. Allow this mixture to
incubate at room temperature
for 5 minutes.
A. Strawberry DNA Extraction
Using a transfer pipette, fill the microcentrifuge tube
with tap water. Close the tube and mix the contents by
inverting the tube or vigorously tapping the bottom of
the tube with your finger. Spin the tube in the
microcentrifuge at high speed for 1 minute.
Decant as much of the water as possible without
disturbing the DNA pellet. Refill the microcentrifuge
tube with tap water and repeat the mixing step. Spin
the tube in the microcentrifuge at high speed for 1
minute.
A. Strawberry DNA Extraction
•
•
•
Remove as much of the water as possible without
disturbing the DNA pellet.
Using a transfer pipette, resuspend the DNA in 1 ml
tap water by taking it up and down in a transfer
pipette repeatedly. [BE CAREFUL]. Transfer to a small
glass cuvette and add 4 ml of tap water. Uniformly
resuspend the blue-stained DNA in the 5 ml solution
by pipetting up and down repeatedly. [BE CAREFUL]
Your stained DNA solution is highly stable and should
be saved for the remainder of the laboratory period.
As part of the standard curve analysis, read and record
the absorbance of the 5 ml DNA-containing solution at
550 nm.
Assignment for Week 1-2
• Complete and turn in the form on p. 7.
• Do Library Assignment 1 tutorial. Libr I Quiz on 9/4.
• Read Chapters 1 to 4 (4 & 5 for week 3). Test 1., 9/11 (Ch. 1-4).
Read Diversity Lab instructions before coming to class on 9/11.
• Finish reading the Scientific Experimentation: Standard Curve
Analysis for DNA Quantitation including Writing Lab Report 1
and data analysis (pp. 12-17).
• Files on How to Write a Scientific/Lab Report are for your
reference. Please follow these useful instructions when you
write the three lab reports.
• Purchase and turn in one pack of AccuScan #815-E or
equivalent by 9/4.
• Have textbook for study. Start the Threaded Discussion
participation.
Thank you for keeping the lab clean,
posting TD and your Facebook
• Test #1. 6:45 – 7:25. Back @ 7:30.
• Turn in signed p. 7 of syllabus.
• Turn in one pack of scantron. Put your name on the
Post-it.
• Go over lab today. 2-3 per group.
• Set up standard curve, measure your DNA sample.
• Enter your group’s standard curve date in Excel in
front.
• See Announcement about Week 2 Assignment.
Standard Curve Preparation
Applications
•
•
•
•
•
Protein/DNA determination
Enzyme assay
Immunoassay/ELISA
Radioimmunoassay
Virtually all clinical assays
A Standard Curve
120
Grades
100
80
60
SD: Standard Deviation
40
20
0
51
2
10
3
15
4
205
Times Wasted in Studying Bio 21 Per Week (hrs)
Series2
n = 352
2 beakers
2 pipet bulbs
5 test tubes
Materials for
tonight’s lab
Test tube rack
1 wax pencil
Kimwipes
5 Cuvettes
1 ml, 5 ml, 10 ml pipets
Spectrophotometer
Materials for
tonight’s lab
2 beakers
1 pipet bulbs
3 pipets
5 test tubes
Test tube rack
1 wax pencil
Spectrophotometer
Intro to Spectrophotometer
p. 2
• Beer’s Law: the concentration of a substance in solution is
directly proportional (linear, p. 2) to the amount of light
absorbed by the solution, and inversely proportional to
the log of the fraction of light transmitted by the solution.
• The incident light is monochromatic (light of a single
wavelength.
P. 23
wavelength
vs. color
UV spec
Radiation
spectroscopy
Light dispersed by a grating into a continuous spectrum of colors.
The wavelength is adjusted by rotating the grating so that a small
selected region of the visible spectrum passes through the exit slit
into the sample.
Light of one wavelength (and color) is passed through a sample of
solution and than illuminates a phototube detector.
The detector produces an electrical signal corresponding to the light
intensity. The magnitude of the signal is displayed on a meter.
Initial [MB] (%)*
Dilution factor
Dilution Procedure
5ml stock * (1 g/L)
+ 45 ml H2O in beaker
Final [MB] (%)
Need 5 ml each
0.1%
Stock
1:10
0.01
1:10
1 ml of 0.1 %
9 ml of H2O
0.001
0.01
1:5 or 2:10
1 ml of 0.1 %
4 ml of H2O
0.002
??
0.003
0.04
0.05
0.06
0.07
0.08
0.09
3:10
0.01 % working
solution
Initial [MB] (%)*
Dilution factor
Dilution Procedure
Final [MB] (%)
Need 5 ml each
0.1%
Stock
1:10
5 ml stock * (0.1 %)
+ 45 ml H2O
0.01 % working solution
0.01
1:10
1 ml of 0.01 %
9 ml of H2O
0.001
0.01
1:5 or 2:10
2 ml of 0.01 %
8 ml of H2O
0.002
0.01
3:10
3 ml of 0.01 %
7 ml of H2O
0.003
0.01
4 ml of 0.01 %
6 ml of H2O
0.004
0.01
5 ml of 0.01 %
5 ml of H2O
0.005
0.01
6 ml of 0.01 %
4 ml of H2O
0.006
0.01
7 ml of 0.01 %
3 ml of H2O
0.007
0.01
8 ml of 0.01 %
2 ml of H2O
0.008
0.01
9 ml of 0.01 %
1 ml of H2O
0.009
[MB] (%)
Absorbance @ 550 nm
0.01
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
1.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
DNA Quantitation
nmnm
550490
@@
Absorbance
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
00
.002
0.02
.004
0.04
.006
0.06
.008
0.08
.001
0.1
0.12
KMnO4
Methylene
BlueConcentration
Concentration (%)
Figure 1. Standard Curve Showing The Relationship Between The Concentration
Of Methylene Blue And Absorbance At 550 nm. Ten different dilutions of
Methylene Blue were prepared by ??? Each solution was read ??? The curve will
be used for ???
Methylene Blue
goes into waste
chemical bucket.
When you are
done,
• Enter your group’s data when done, they will be
posted tomorrow.
• Clean up everything that you have used on your
bench
• Wash and invert all test tubes
• Do not forget to take the cuvette out of the spec
Sample well
For 0% T, empty
Wavelength, use 550 nm
For 100% T, use water
Calibration
• Cuvette with water: 100%
transmission (0% absorption)
• Nothing in sample slot: 0%
transmission (100% absorption)
Be absolutely certain about calibration, reading
absorbance, using significant numbers, accuracy, and
precision before doing experiment
Reading
Reading is 0.239
If reading is >1, the reading can only
be e.g., 1.13 not 1.134, why?
INCORRECT