Biochemistry Lab I

Biochemistry Laboratory I

Syllabus

Required Textbooks:

A Short Guide to Writing About Biology, 5th Ed. 2004 Jan A. Pechenik
At The Bench: A Laboratory Navigator BARKER, CSH

Click here to see the help files
and tutorials
- Tegrity Files
- List of Protocols

Date	Required viewing	Pre-Lab Quiz	Experiment Handout	Description
26/28 Aug	Teg 1&2 Protocol 1 How specs work and Wiki	Quiz 1	Lab Introduction, Pipetting, Spectroscopy, and lab book	Intro to class expectations, safety and grading. Will start with a pipetting exercise
2/4 Sept	Teg 3 Using the pH Meter	None	Lab Math, pH meters, buffer preparation	Practice with typical biochemical math problems and techniques
9/11 Sept		Quiz 2	DNA Purification and Quantification NEW!!! - The handout above is correct accept for the plasmid purification. For the actual protocol, use the instruction manual for the kit (click here). All else is as written. Read manual background before lab.	Purification of fusion MDH-GFP-His plasmid DNA
			Turn in Lab Book -Sept 12
Sept 16/18	Teg 4 Pouring DNA Gels Protocols 4&5	Quiz 3	DNA Transformation and DNA Electrophoresis	Insertion of purified plasmid and analysis
Sept 23/25			Writing Scientifically - Bring the printouts from the links below to class. Required Reading Writing about Biology pages: Coming SOON! - JBC Example - MGH Paper Guidelines - JBC Instructions to Authors	Highly Recommended Reading! From Dr Caprette, RIce University - Common Writing Errors - Fun Riting Wrules.
Nov 30 Oct 2	Tegs on equipment and standard curves.	No Quiz Printout and read Chromatography and the purification handouts.	Chromatography and Protein Analysis Handout (Tutorial assn) You MUST complete the tutorial as described in the handout (link above) on your own. The assignment is due 6 Oct! On-Line Purification Tutorial	Background on purification and using equipment for protein purification
Oct 7/9	Protocols on purifications and protein/MGH assay	Quiz 4 - Write a short but specific plan for purification. NOT the same as the homework from the tutorial!	Purification I Purification Handout See links below for protocols and specifics on using equipment and assays.	Choose two of four different chromagraphic techniques to purify your expressed fusion protien
			Turn in Lab Book -Oct 10
Oct 14/16		DO NOT FORGET the writing deadlines. No reminder will be given...	Purification II
Oct 21/23			Purfication III
Oct 28/30			Purification IV
Nov 4/6			Purification V
Nov 11/13		Quiz 5	SDS PAGE / Western Blot Turn in Lab Book -Nov 14	Analysis of protein purification and antibody recognition
Nov 18/20		Quiz 6	Develop Blot Instructions for Developing Blots Enzymatic Analysis Enzyme Assay Background	Finish Blot and Introduction to Enzymatic Analysis.
Nov 27/29			Off - Thanksgiving
Dec 2/4		Quiz 7	Enzymatic Analysis	Determine the kinetic characteristics of MDH and investigate inhibition - Plan and execute your own experiment
Dec 12 3:00			Lab Practical / Check Out / Turn in lab book	Your presence is required for a passing grade!

Deadlines:

Lab Book Turn in 12 Sept, 10 Oct and 14 Nov – Turn in book to Chem Dept Office
3 Oct Draft of Intro and Methods for DNA Purification and Transformation
20 Oct Draft of Results and Conclusions for DNA Purification and first purification
14 Nov Draft of figure w/ legend for DNA Gel, Standard Curve and First Chromatograph
26 Nov Draft of Full Manuscript / Paper

Tegrity Files - Help w/Tegrity	Links & Manuals	Protocols
1 - Using LTS Pipets for the experienced user	Rainnin Pipet Manual (For reference only) Using Pipets Ranin web link	1 - Sipper set-up for the Beckman Spectrophotometers Protocol
2 - Using The Beckman DU Spectrophotometer	How a Beckman Spectrophotometer Works Spectroscopy from Wikipedia	2 -Pouring and Running a DNA Minigel / DNA Quantitation (UV Vis and Dye based methods) Protocol
3 - Using the pH meter	Preping and Running DNA Gels (Colorado State) Using a pH meter Wiki Background	3 - How to use the Kodak Camera System
4 - Pouring DNA Gels	Chromatography-Online - A good site for advance information. Look at the principles and practices link and other links for more insight.	4 - Bacterial Culturing Protocol
5 - Setting up column adaptor	Protein Chemist - A very source of info on each purification step, buffer calculators, gels and antibodies. Spend some time here, it will help you!	5 - Transforming Comp Cells Protocol
6 - Setting up pump, column and fraction collector	Protease Inhibitor Use page 1 page 2 Protein Assay - a very nice and complete review	6 - Ammonium Sulfate ppt Protocol
7 - Using a gradient maker	Calculating pI values Expasy - ProtParam tool for calculating pI and size. Scripps page molbiol tools page	7 - Ion Exchange Purification Protocol
8 - Generating standard curves	Ammonium Sulfate Precipitation Table Ammonium Sulfate Precipitation Calculator	8 - His Tag Affinity Purification Protocol
9 - Protein Purification Overview	BioRad Column Flow Adaptor Manual	9 - Reactive Dye Affinity Purification Protocol
10 - Assembly of the SDS PAGE apparatus	MGH Protein & DNA Sequence (word doc) LOOK FOR THE FILE IN THE DOWNLOADS! IT MIGHT NOT SHOW UP UNTIL YOU LOOK FOR IT. If all else fails, under the finder, go to the file pulldown window and do a find "mgh protein" Be certain to use the amino acid sequence NOT the DNA sequence for this program. The DNA sequence gives the WRONG numbers.	10 - Size Exclusion Purification Protocol
11 - Assembly and westernblot instruction	Peristaltic Pump Manual	11 - Hydrophobic Interaction Chromatography Protocol
	Ultrafiltration (Centriprep) Manual	12 - Purification Planning Check Sheet
		13 - Peristalic Pump Instructions
		14 - Column Adaptor Instructions
		15 - Fraction Collector Instructions Pharmacia Ready Frac 100 &200 BioRad Model 2110
		16 - pH Meter Instructions
		17 - Dialysis Instructions / Protocol
		18 - Ultrafiltration Protocol
		19 - Protein Assay Protocol - 96 Well Plate Protein Assay (USE THIS ONE) - Plate blank - use for planning your assays
		20 - GFP Flourescent Assay Protocol (Updated 16 Oct)
		21 - Preparing A Purification Table
		22 -SDS PAGE Gel Instructions / Protocol
		23 - Westernblot Protocol
	For more research protocols / guides - go to the bottom of my research page

	Need Help For Viewing Tegrity Files?

Hints on two axis graphs

Create three columns the first column will be elution volume, the second two will be your intensity and protein data.
Using your cursor, highlight all three columns
click on the graphing wizzard
click on the "custom tab"
Scroll down until you get to "Lines on 2 Axis"and click on that
there will be three series in the dialoge window, select each series and then click in the Values box - look at your spread sheet. When the x-axis data is highlighted, click on the series in the series box and remove it.
Then on the bottom of that dialoge box, enter in your X axis.
Don't forget that this is the place to label each of your axis as well.

HINT for Error Bars - USE OLD EXCEL Program!!! Excel 2008 does not have the plug in for error bars.

Rice Biochemistry

A few GFP links .
Clonetech - you may recognize this one

More info you've seen
Silke Jonda's PPS2 project