Understanding DNA and Gene Cloning:
A Guide for the Curious
by Karl Drlcia

ISBN: 0471434167

Reading the Story in DNA
by Lindell Bromham

ISBN: 0521870704

• Read the Paul Nurse paper "The Great Ideas of Biology" Clin. Med. 3(6): 560-568. (PDF)

• Handout (PDF)
• Laboratory basics:
  • pipetting
  • “cell management” – sterile technique and plating
  • biological spectroscopy using a spectrophotometer

• Brief summary in your lab notebooks of each of the three laboratory exercises.

• Read the brief manual (PDF) we have written on micromanager, the software we will be using to run the fluorescence microscopes. Also, you will find it useful to review the Matlab tutorial.

• We have a variety of prepared samples we want you to look at: resolution target, marine diatoms, fixed and fluorescently labeled eukaryotic cells, fluorescent E. coli, pond sample. In each case, you will need to take pictures which will be turned in to us (with appropriate scale bars).
  • resolution target – picture with the 100x objective
    
  • fluorescent eukaryotic cells – picture in 3 fluorescence channels
    
  • E. coli – phase image and fluorescence image
    
  • Pond water from Caltech ponds – what do you see?
  • marine diatoms – single nice picture (if you have time)

• Assignment 1 (PDF)
• Please see the following two Matlab handouts for assistance doing the Matlab portion of the homework:
  • Calibration tutorial
  • Fluorescence merging tutorial

• Reading:
  • Whitman, W., et al. "Prokaryotes: the Unseen Majority" PNAS, 1998. 95: p. 6578-83 (PDF, Caltech only)
  • Venter, J. C., et al. ""Environmental Genome Shotgun Sequencing of the Sargasso Sea" Science, 2004. 304: p. 66-74 (PDF, Caltech only)

• Protocols for Session 1 (PDF)
  • Pond water collection
  • Sample centrifugation & storage
• Journal club #1

• Reading:
  • Water sample DNA extraction protocol (PDF)

• Protocols for Session 2
  • DNA Extraction
  • Spectrophotometry of extracted DNA

• Drlica reading: pp. 40-47, 153-15 (these are the sections on DNA replication and PCR)
• PCR prelab / solutions (PDF)

• Protocol for session 1 (PCR)

• Examination of:
  • Carolina samples
  • Pond water samples (unstained and DAPI/SYBR stained)
  • E. coli (unstained and DAPI/SYBR stained)

• van den Broek, et al. "Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI" Nucleic Acids Research, 2006. 34(1) p. 167-174 (PDF, Caltech only)
• Week 4 Prelab assignment
• Drlica: p. 118-138

• Restriction digest (handout, PDF)
• Set-up for single molecule restriction digest

• Gel analysis of bulk restriction digest (handout, PDF)
• Continuation of single molecule restriction digest

• Assignment for Monday, April 27
  
  
  • Read pgs. 185-215 of "Reading the Story in DNA". We will be discussing this chapter in class on Monday evening in journal club format. (This assignment is also listed below as reading).
  • Week 5 prelab
    
    
• Assignment for Tuesday, April 28
  
  
• Provide three microscopy images of different samples with at least one of them coming from the pond water (from Week 3, Day 2). Your other samples can be the Carolina samples. In each case, please make sure to include a scale bar and a few sentences on how you are sure that the scale bar is correct. In addition, in each case provide a one paragraph (max.) description of the organism. In your description, try to focus on those features of the organism that you think might be relevant to exploring the evolutionary relatedness of your organism to other organisms. What we are driving at is that we want you to come up with various characters that you think are possibly relevant to classification.

• Read pgs. 185-215 of "Reading the Story in DNA". We will be discussing this chapter in class on Monday evening in journal club format.
• Week 5 prelab assignment (PDF)

• Week 5 Lab handout (PDF)

• Week 5 Lab handout (PDF)

• TPM Assignment (PDF) due Tuesday, May 5

• Philip Hugenholtz "Exploring prokaryotic diversity in the genomic era" Genome Biology 2002, 3(2):reviews0003.1–0003.8 (PDF, Caltech only)
  
  
• Week 6 prelab assignment

• Week 6 Lab handout (PDF)

• Turn in a write up that shows your bacterial growth data and in which you obtain the cell doubling time. Your work should be turned in with proper figure captions, discussions of what you did and what you learned, some concluding thoughts, etc.

• Jacques Monod "The growth of bacterial cultures" Annual Reviews Microbiology. 1949. 3:371-394 (PDF, Caltech only)

• Single cell microscopy (group 1) - Protocol, (PDF)

• Single cell microscopy assigment (PDF)

• Picking colonies, single cell microscopy (group 2) - Protocol (PDF)

• Single cell microscopy assigment (PDF)

• Rosenfeld, et al., "Gene Regulation at the Single-Cell Level, "Science. 2005. 307:1962-1965

• DNA Extraction
• Spectrophotometry of extracted DNA

• DNA Extraction
• Spectrophotometry of extracted DNA