Roe Lab Protocols

• Roe Lab Protocol Book

• Gel Preparation and ABI 377 XL Protocols - updated 3-9-98

Most recent versions of our protocols

• Emulsion Breaking Using Centrifugation

• Longer Read Lengths on the 454 GS20

• Download the Cooling System PowerPoint file

• Download the Control Beads at 84 Flows PowerPoint file

• Cleaning the PicoPlate for Re-use

• Download the Cleaning the PTP quicktime movie

• 384-well DNA Sequencing Template Picking, Growth and Isolation using the QPix Colony Picker, HiGro Oxygenated Shaker Incubator, Zymark Mini-Staccato, and Velocity 11 V-Prep robots - New 03-03-03

• BAC DNA Isolation from 200 ml Culture by the Cleared Lysate Method Followed by Double Acetate Precipitation - Updated 01-27-02

• BAC and Fosmid DNA Isolation using 96 deep well, blocks, pipetting on the Hydra 96, and growth in the Floor Shaker for end sequencing - Updated 12-12-07

• 384 well DNA sequence reaction pipetting from the 96 well pre-dispensed, diluted, BigDye V3.0 and V3.1 or ET mix plates to 384 well viper plates Updated 01-24-06

Improved Closure Protocols

• Protocol for Enhancing PCR of Very Difficult Regions - Updated 02-14-02

• Protocol for Sequencing Very Difficult Regions - Updated 02-14-02

• Gap Closure using the Amersham Phi29 Sequence Finishing Kits for DNA Amplification Prior to Sequencing - Updated 05-26-05

• Gradual vs Standard PCR and Colony PCR Protocols - Updated 02-26-06

• Sequencing Reaction Cleanup in 384 Well Sequencing Reaction Plates - Updated 12-11-02

• 384 well thermocycler plate cleaning for re-use - New 12-31-02

• Lander-Waterman Calculations updated for 454 GS-20 100 & 200 base reads:

• Lander-Waterman Tables 1, 2, and 3
  Table1:The probability a base is not sequenced,
  Table2:TotalGapLength vs % Sequenced
  Table3: Number of Sequence Reads for x-fold Coverage

• Lander-Waterman Table 4:Number of Gaps

• Lander-Waterman Summary Table for 100 and 200 base reads

• Zebrafish whole mount in situ hybridization protocols adapted from zfin.org

• 384-well DNA Sequencing Template Picking, Growth and Isolation using the Flexys Colony Picker, HiGro Oxygenated Shaker Incubator and Hydra 96 Automated Pipettor with Moving Stage

• Manual Large Scale Plasmid, Cosmid, BAC, PAC, and Fosmid DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation

• Isolation of Cosmid, BACs, PACs and Fosmid DNA using 96 deep well blocks, pipetting on the Hydra 96, and growth in the HiGro

• DNA Sequencing Template Picking, Growth and Isolation using the Flexys Colony Picker, HiGro Oxygenated Shaker Incubator and Hydra 96.

• Making and using predispensed Big-Dye DNA sequencing pre-mixes.

• Ethanol precipitation of DNA sequencing reactions

• Uniplex PCR-based Sequencing

• MultiPlex PCR-based Sequencing

• Cloning MultiPlex PCR-based Products for DNA Sequencing in pUC-based Vectors

• Sequencing off Plasmid Templates with ABI Bi g Dye Terminators

• Sequencing off Cosmid, BAC, PAC, and Fosmid Templates with ABI Big Dye Terminators

• ET Terminator Reaction Protocol for MegaBACE

• LICOR Terminator Sequencing Protocol

• LICOR Tracking and Post-Sequence Processing

• Reduced Volume AmpliTaq-FS DNA Polymerase Cycle Sequencing Reactions on the Robbins Hydra96 with Computer Driven Stage

• Isolating Double-Stranded Sequencing Templates on the Biomek and Hydra via Single Acetate Cleared Lysate Proc.

• Isolating Double-Stranded Sequencing Templates on the Biomek and Hydra via Double Acetate Cleared Lysate Proc.

• Manual 96 well ds template isolation for sequencing with fluorescent terminators

• Automated Biomek 2000-based Double-Stranded DNA Sequencing Template Isolation

• List of companies that supply pre-made gel mixes, replacement components for the ABI Fluorescent sequencer and the nebulizers for shearing DNA

• Solving the Red Rain problem on the 377 48cm well to read gels

• Finding Repeat Sequences with Jeremy Parsons Micropeats program

• Poisson Calculations - Lander/Waterman

• Sequencing off Bacterial Genomic DNA with ABI Big Dye Terminators

• Link to Skip Garner's MerMade Web Site