Protocols Used in the Roe Laboratory

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Roe Lab Protocol Book

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


Most recent versions of our protocols

• Modifications to 454 protocols
  • Amplification primer study for sequencing Amplicons on the FLX with the long read (plus) upgrade - Updated 4-14-2012
  • Download our paper describing our Methods for GeneratingShotgun and Mixed Shotgun/Paired End Libraries for the 454 Sequencer in Current Protocols In Genetics 2009. - Updated 3-8-2012
  • Download our latest emPCR protocol the Titanium chemistry. - Updated 3-8-2012
  • Download our latest semi-automated library protocol using the Zymark SciClone robot. - Updated 06-18-2009
  • Longer Read Lengths on the 454/Roche GS-FLX - New 10-01-2008
  • Robotic Library Preparation on the Caliper/Zymark SciClone
    • View a Quicktime Movie showing the Introduction and Methods portion of our Automation protocols - New 10-01-2008
    • Building the Enzyme Chiller and Magnetic Separator - New 10-01-2008
    • View a Quicktime Movie showing our Zymark Caliper SciClone robot preparing libraries for the 454 - New 10-30-2008
  • BAC growth and Hydra-based isolation for MID-labeling and pooling on the 454 - New 12-15-2009
  • Emulsion Breaking Using Centrifugation
  • Cleaning the PicoPlate for Re-use
  • Longer Read Lengths on the 454 GS20
• 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

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  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

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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:

1. 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
2. Lander-Waterman Table 4:Number of Gaps
3. Lander-Waterman Summary Table for 100 and 200 base reads

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

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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