Thursday, December 12, 2013

Lab Orientation, Safety, & Pipetting

Lab Orientation, Safety, & Pipetting.pptx
Puffenbarger, R. 2011. Understanding Solution Chemistry the “Southern Way” With Sweet Tea. Pages 117-126, in Tested Studies for Laboratory Teaching, Volume 32 (K. McMahon, Editor). Proceedings of the 32nd Conference of the Association for Biology Laboratory Education (ABLE), 445 pages. http://www.ableweb.org/volumes/vol-32/v32reprint.php?ch=10

Introduction
The pipette is designed to measure and transfer very s,mall volumes of liquid with accuracy and precision. Accuracy refers to the margin of error between the dialed volume and the volume actually delivered by the instrument. Precision refers to the ability of the instrument to deliver a given dialed volume repeatedly. The pipette is far more accurate than glass or plastic serological pipettes especially when volumes less than a milliliter (ml) are desired. 

Parts of the Pipette 



Figure 1. External parts of the pipette
-Plunger button
-Tip ejector button
-Volume adjustment dial
-Volume indicator
-Shaft
-Tip ejector
-Disposable tip

A plastic tip placed on the bottom of the shaft to prevent cross-contamination between samples and to prevent liquid from being pulled into the shaft.
Volume is adjusted by turning the volume adjustment knob or dial.
A pipette function by air displacement. When the plunger is depressed, all air is expelled from inside the shaft by a metal rod or piston, creating a vacuum. As the plunger is released, the rod retract to a point set by the volume dial and the liquid sample flows into the plastic disposable tip replacing the air that was displaced form the shaft. The rod is controlled by a spring mechanism. Dialing the pipette above the maximum volume rage of the pipette distorts the spring leading to inaccurate measurements. If the pipette dial is turned too far past the max volume for the unit the spring can snap entirely. 

Reading the scales on the pipette 
For the P10 and the P20, the last digit in the window is a decimal. The P200 has no decimal places. On the P1000 the "ones" place or the final digit is the number 1000 is not represent on the scale. 




Choosing the correct pipette for the task


Pipettor Theoretical Range (ml) Recommended Range (ml)
P10 0-10 1-10
P20 0-20 2-20
P200 0-200 20-200
P1000

0-1000

100-1000

Operating the pipettor


1.) Set the volume
2.) Attach a disposable tip
3.) Load the sample
4.) Expel the sample


Learning Objectives: 
1. Defining molarity
2. Dilute solution by on fold change
3. Use the C1V1=C2V2 equation to create solution of the proper molarity using give stock solutions
4. Determine how much of a compound is needed to make a solution of the desired molarity.
5. Determine how to make a solution described in therm of % w/v or % v/v

Background:
Expressing solution concentrations:
The Percent (%) Solution:
Molarity Concentrations:
Converting Between Units:
Lab Exercises/Procedure:

Reference to Department of Biology and Biochemistry Fall 2013, BCHS 3201 Biochemistry 1 Laboratory Manual, University of Houston.

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Title: Making Solutions and Determining Sugar Tasting Threshold
Preparing and Determining molar concentrations

Prelab
a.) Scientific concept(s)

  • Learn 3 ways concentration are expressed. X Dilutions, Molarity and percent
  • Understand what molarity and % mean in terms of solution chemistry.
b.) Laboratory techniques
  • Preparing a solution by dissolving a solid solute in a solvent.
c.) Objective (goal)
  • In this lab we will determine the lowest molar concentration (threshold) of sugar that the class can taste

Results
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This graph illustrates the sugar concentration (M) and the amount of students that could taste said concentration. The lowest threshold is .01M

Discussion/PostLab

#2) *note the placebo effect

BCHS3201 Syllabus Fall 2013

BIOCHEMISTRY LABORATORY I
BCHS 3201
                                   Fall 2013

Lab Coordinator:  Dr. Donna Pattison
Office:  Room 108C, Science Teaching Laboratories Building
Phone Number:  713-743-2642
e-mail:  dpattison@uh.edu
Office hours:  Monday and Wednesday 9-10 am or by appointment

Teaching Assistant:________________________________
TA e-mail:_______________________________________

Text:  Purchased from Research Stores on the second floor of the Science Building.  A biochemistry textbook will be very helpful to you as you write your lab reports.  The Voet, Voet, Pratt used for BCHS3304 is recommended.

A calculator may be helpful.  Lab coats and goggles are REQUIRED.  You will also need a tear-out lab notebook with duplicate pages. Goggles, lab coats and notebooks are available for purchase at Research Stores on the second floor of the Science Building. 

Course Description:  This course reviews the nature of the chemical constituents of living organisms, including nucleic acids and protein.  Students will gain hands-on experience in common lab techniques used to study nucleic acids, proteins and enzyme activity.

Objectives:
Students will:
1) demonstrate basic skills in laboratory math.
2) demonstrate the ability to use common laboratory equipment such as centrifuges,
     pipettors, chromatography columns, and electrophoresis equipment.
3)  be able to critically analyze and explain their experimental results.
4)  apply the scientific method and their knowledge of biochemistry to design and carry
     out experiments.

Week                                      Lab
August 26-29                           Lab Orientation and Safety; Pipet Techniques

September 2-5                         Making Solutions and Determining Your Sugar Tasting
                                                Threshold; Graphing in Excel (no class Monday,
                                                September 2)

September 9-12                       Plasmid Isolation
Quiz 1
                                                           
September 16-19                     Restriction Digests of DNA and Gel Electrophoresis
Using the Computer in Biochemical Research I:  BLAST Search and Sequence Alignment (Homework due)

September 23-26                     Identification of Serum Glycoprotein by Polyacrylamide Gel Electrophoresis and Western Blotting
Using the Computer in Biochemical Research II:  Restriction Digests (Homework due)
                                   
September 30-October 3         Fold-It:  Computer Assisted Protein Folding Predictions; Quiz 2
                                               
October 7-10                           Protein Crystallization; Crystallography Pre-Lab Homework Assignment Due; Quiz 3
                                                                                                           
October 14-17                         Size Exclusion Chromatography; Literature Search Homework due

October 21-24                         Complete Protein Crystallization lab; Writing Workshop                       
October 28-31                         Chloroplast Extraction and the Hill Reaction:  The
                                                Biochemistry of Photosynthesis
                                               
November 4-7                         Crystallography Paper Rough Draft due; Peer review
                                                editing session; Quiz 4

November 11-14                     Enzyme Kinetics:  Turnip Peroxidase
                                                Protein Crystallization Manuscript due

November 18-21                     Enzyme Kinetics:  Turnip Peroxidase (continued)

November 25-28                     No labs.  Thanksgiving Holiday

December 2-5                          Last lab report due; Quiz 5

                       
                                               
*****Please note that the above schedule is tentative and is subject to change based on the availability of materials from our suppliers.  Your teaching assistants will inform you of any changes.
                                               



Class Policies

Laboratory Attire:  Lab coats are required.  You will not be allowed to attend the lab without a lab coat.  Safety goggles will be required for certain labs.  Gloves will be provided as needed.  Please inform your instructor if you are allergic to latex.   

Guesting:  If you miss a lab for an excused reason, inform your
TA as soon as possible.  Make arrangements with another TA (see schedule posted on classroom door and on Blackboard) to guest in their lab.  You may guest up to two times in a semester.  Please contact your regular TA and the TA with whom you will be guesting for permission.  You do NOT need to contact Dr. Pattison for permission.  No more than 5 students will be permitted to guest in a single section.  You are also required to fill out the guesting form (located on the TA bench at the front of the room) when you arrive for the section in which you are guesting.  There are no labs on Friday. You will take the quiz for the lab section you are visiting. 

Absences: Attendance is MANDATORY. As the main point of a laboratory course is to gain hands-on experience in lab techniques, if you fail to attend more than two labs, you will automatically fail the course unless you provide evidence of extenuating circumstances that are excused under university policies.  It is YOUR responsibility to deal with attendance issues PROMPTLY.  If there are extenuating circumstances and you have already guested twice, you must speak with both your TA AND Dr. Pattison. 

Late work: Failure to hand in 2 or more lab reports will result in an F even if you attended the lab.  Lab reports are due at the start of class.  Reports will be subjected to a ten percent loss in points for failure to turn the report in at the start of class and for each day beyond the due date. Lab reports that are more than 7 days late will receive a zero. 

Arriving Late: You are expected to arrive in class on time.  You will not receive additional time to complete a quiz if you are late.  Points will be deducted from your participation grade for tardiness.  If you are more than 15 minutes late, you are considered absent and will not be allowed to participate in the lab.    You will need to guest in another lab to obtain credit for that lab. 

Incomplete Grade Policy:  A temporary grade of “I” can be assigned by the instructor when a student is currently (A) passing a course or (b) still has a reasonable chance of passing in the judgment of the instructor, but for non-academic reasons beyond their control have not completed a relatively small part of all course requirements. After the student and the instructor agree that the student shall receive an “I” grade, an “Incomplete Grade Agreement” form must be completed and filed with the Office of Undergraduate Affairs (124F, Science Building).   Further information on “I” grades can be found at http://www.uh.edu/academics/catalog/general/acade2.html#grades.

Students with Special Needs:  The Americans with Disabilities Act of 1990 requires that universities make reasonable accommodations to persons with disabilities as defined in the act.  Students who feel they need assistance as defined by the guidelines set forth in the act should contact the lab coordinator, Dr. Pattison, to discuss appropriate arrangements. 

Important Dates:
September 11:  Last day to drop class without a grade
November 1:  Last day to drop class with a “W”

Grades                                   
Lab Activities and Reports (45%)
Pipet technique (No lab report due)     
Making Solutions/Sugar Sensing                                                                       
DNA Plasmid Purification
DNA Restriction Digests
Polyacrylamide Gels
Fold-It
Size Exclusion Chromatography
Hill Reaction                                                                                        
Enzyme Kinetics
                                                           
Journal Style Lab Manuscript (15%)                                               
Crystallization

Quizzes (5) (30%)                                                      

Homework (4) (7%)
Literature Search
Using the Computer in Biochemical Research I:  BLAST Search/Sequence Alignment
Using the Computer in Biochemical Research II:  Restriction Digests
Crystallography Pre-Lab Homework
                                               
Participation   (3%)   
                                                                            
Grade Assignments                       
%                                 Letter Grade
92.0 – 100%                A
90.0 -  91.9%              A-                      
88.0 - 89.9%               B+                      
82.0 – 87.9%               B                  
80.0 – 81.9%               B-                     
78.0 – 79.9%               C+
72.0 – 77.9%               C 
70.0 – 71.9%               C-                    
68.0 – 69.9%               D+ 
62.0 – 67.9%               D
60.0 – 61.9%               D-

≤59.9%                        F