Functional Genomics Research Stream. Lecture: March 31, 2009 Topics: Continued Gel Electrophoresis, Nucleic Acid Preps
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1 Functional Genomics Research Stream Lecture: March 31, 2009 Topics: Continued Gel Electrophoresis, Nucleic Acid Preps
2 Agenda Student Meetings Fall Course Information DNA, RNA Preps Review Gel Electrophoresis Gel Visualization Assignment Nine Progress
3 Student Meetings This Week Sign Up Sheet 15 Minute Meetings Bring: Laboratory Notebook Bring: Assignment Nine
4 Fall Research Course: The Schedule
5 Fall Research Course: The Details Far Fewer Assignments (if any) Independent Research Computational Training, Bioinformatics Degree Plan Credit (one of four research courses required) Publication Potential Research Reference Letters
6 DNA & RNA Preps 1. Liquid Culture 2. Cell Spin Down, Re-suspension 3. Cell Lysis 4. Phase Separation 5. Retrieve Aqueous Phase 6. Precipitation of Nucleic Acid 7. Pellet Nucleic Acid, Re-suspend
7 The Steps to Prep culture cell spin down resuspend cell lysis phase separation aqueous phase precipitation pellet
8 Phase Separation proteins nucleic acids aqueous phase Spin Real Fast interphase organic phase Cell Lysate +Phenol Phase Separated
9 Phase Separation: Caution proteins nucleic acids aqueous phase Do Not Get Greedy interphase organic phase
10 Gel Electrophoresis Nucleic acids are positively or negatively charged molecules? Charged molecules move toward or away from opposite charge?
11 University Leicester in Education
12 University Leicester in Education
13 Gels: Top View
14 Gels: Side View - +
15 Gels: Loading & Running
16 Gels: Result
17 Gels: Visualization Use a gel to tell us something diagnostically. Gels can also be used for sample preparation. Current Protocols Essential Laboratory Techniques, Wiley
18 NanoDrop ND-1000 Quantity & Quality Evaluation Possible
19 Work This Week
20 Reagent & Solution Making Recommended: Making all reagents at ~ 12 ml volume directly in 15 ml conical tube. 1. Measure dry reagents. 2. Add dry reagents to tube. 3. Bring to volume in tube. 4. Mix well, vortex, ph if needed.
21 Review: Yeast Growth colony dilute to ~0.2 Streaked Plate Overnight Culture overnight Large Culture during day
22 Review: Growth Curve OD600 ~ 0.80 OD600 ~ 0.2 Current Protocols in Essential Laboratory Skills
23 Doubling Times Yeast: ~ 2 hours Let us say you start a culture OD When OD hours later? When OD hours later? When OD hours later? When OD hours later?
24 Why Snap Freeze? If cells removed from good growth conditions, transcription changes. We are not interested in measuring this response. We wish to grow cells and then capture a moment in time.
25 Two Weeks Left (Assignment Nine) Many of you burned a week: Not a Good Idea Easiest Work: Cell Plating & Growth - Done Before Hardest Work: DNA & RNA Preparation, PCR Staff Crazy Factor Helping You Finish: Zero Percent
26 Issue One: Cleaning & Safety Lab was left a mess on Thursday (March 26). Open beaker of concentrated HCl left out. I am not the morning janitor, neither are any of the research staff. Lab citizenship grade now managed quietly and without any warning.
27 Issue Two: Waste 10 students started overnight culture on Thursday (March 26). Of 10, only 1 student read ahead and had any hope of doing large culture next day. Thus, 9 overnight cultures wasted. (wasted time, resources, space) Read ahead and think, ask questions.
28 Issue Three: Laboratory Notebooks Everyone is doing a good job. Everyone is doing too good of a job. While you work, not after you work. (especially for upcoming DNA, RNA) Temporal flow; not a report.
29 Change Requested Notebooks open on bench. (good way to claim a bench space) More work in notebooks. (while working) Less report format, more a log of your laboratory activity.
30 Questions?
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