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Step 1: Creating dsDNA encoding the part for cloning Two variations on preparing a dsDNA fragment with the proper restriction sites and Golden Gate overhangs are provided...

SeanLeonard 14 Sep 2017

Upon publication, this page will be updated with a full description of the BTK, parts list, and overhang types. SeanLeonard 14 Sep 2017

Back to Golden Gate Protocols Designing a new part Golden Gate Assembly Step 1: Creating dsDNA encoding the part for cloning Two variations on preparing a dsDNA...

SeanLeonard 14 Sep 2017

SeanLeonard 14 Sep 2017

The Arabinose (Ara) Genetic Marker Background The Escherichia coli B strain REL606 has a mutation in the araA gene that renders it unable to utilize the sugar...

Quick 3hr Antibiotic Rescue Verification Overview This short protocol describes a simple same day verification of antibiotic removal/`rescue` from counter selection...

Transforming Acinetobacter baylyi ADP1 About A. baylyi ADP1 Acinetobacter baylyi ADP1 is a naturally competent bacteria with enormous potential for genome engineering...

Purifying 6xHis Tagged Proteins from E. Coli by Immobilized Metal Affinity Chromatograpy (IMAC) under Native Conditions SUPPLIES: Equipment: Nutator or Rocking...

Links to Product Manuals Molecular biology NEB DNA Polymerase HF DNA polymerase solution mix DNA Ligase DNA Ligase I, RNase free PNK Assembly Master Mix...

Veracode Golden Gate Genotyping How to process data from RTSF 1 Open GenomeStudio. 1 Create a new genotyping project. 1 Choose the option to `Load sample...

Total Alkaline Digest of Embedded RNA Linkages Materials 0.2 N Sodium Hydroxide (NaOH) Molarity and Normality are related by N nM For example...

Targeted PCR and Sanger Resequencing of Mutations Page in need of additional improvements. Descriptions here are particularly tailored for resequencing of evolved...

topA Gene Sequencing The topA gene coordinates are 1329420 1332017. The topA mutation in the Ara 1 long term line is 1329516:C T. primer who coords...

Pulsed Field Gel Electrophoresis Mapping Purpose: To validate mutations predicted from whole genome re sequencing and possibly discover rearrangements through repetitive...

Primer Extension or Oligo Overlap Extension To stitch together large DNA templates from oligonucleotide fragments. Using Klenow Fragment (3 5 exo...

Population Genetics Long Term Daily Procedure Supplies 1 13 x 50 ml flasks filled with 9.9 ml of DM500 (DM0 supplemented with 0.05% glucose). 1 12 x tetrazolium...

Polyacrylamide Gel Electrophoresis Our gel rigs and supplies are from Scientific. The Diagnostics Website has very helpful background on RNA/DNA polyacrylamide gels...

Petri Dish Patch Templates The Full Template is for patching as many colonies as possible per petri plate. The 96 Well Format is for patching 48 colonies per plate...

Overlap PCR Background Before attempting this somewhat advanced PCR technique, be sure to read the PCR protocol and check out a reference describing PCR theory...

Strains Deleterious Mutations (UV Mutation Accumulation) Strain Fitness Ara relative to REL607 Marker Fitness Ara relative to Ara #8211; Designation...

Label Templates Templates for printing labels...

Major version notes This is a protocol based on Kapa LTP Preparation Kit manual KR0453 v3.13 The main difference centers around the use of 1/2 volume in each reaction...

Genome Minimization Growth / Death Assays In order to be able to compare parameters uniformly between evolved and ancestor strains, all growth in these tests is done...

Protocol for Determining Mutation Rate to Phage (T5) resistance Day 2: Prepare Media/Revive/Titer Phage 1. If necessary, revive the frozen stock of each strain...

Profiling Ribose Operon Deletions Day 1: Search strain databank for desired population. 1. Login to lab website, open lab databases. 1. Search under `strains...

Competition Assays for Evolvability Lines Serial transfer of 3.5 ...

Materials Description Cat # Price Qiagen RNeasy Protect Bacteria Mini Kit (50) 74524 $386.00 Invitrogen Superscript Plus Indirect cDNA Labeling...

ELUTION of OLIGOS FROM PAGE GEL Crush Soak Eluting DNA/RNA from PAGE or denaturing PAGE Materials Crush Soak Buffer (CSB) 200mM NaCl , 10 mM tris HCl (pH 7....

Changing Environment Long Term General Procedures Inoculating Tubes To start an experiment, obtain 98 test tubes. Split the test tubes into two racks, 49 in each...

Mutation Rates from Genome Resequencing Motivation: You have re sequenced several genomes after a mutation accumulation or adaptive evolution experiment. How do...

ASKA Collection Evolution Experiment Starting Lines 1 Revive each strain by inoculating a scrape of frozen culture from a well of a microplate into 3 ml of 0.1x...

Using Emulsions to Select by Yield Background Evolution in suspension culture proceeds by selecting for those strains that grow most rapidly, quickly depleting the...

Using the Deep 96 Well Pin Tool The 96 well pin tool can be used to transfer long term evolution experiments and to make dilutions when plating many samples. Although...

The Checklist for Plating Competitions Before plating, it is important to transfer and mix correctly from the competition plates. The following are guidelines...

The Checklist for Plating Competitions Before plating, it is important to transfer and mix correctly from the competition plates. The following are guidelines...

Packaging a Tool for Release You need these environmental variables set to work with the current CVS setup: export CVSROOT `:ext:local@barricklab.org:/bliss/cvs` export...

Lambda Red Protocol Lambda Red Plasmids These plasmids are available as part of the Lambda Red disruption kit from the E. coli Genetic Stock Center. pKD...

Gene replacement using pKOV vector Before beginning part 1: design primers 1 Insert should be ~1kb with approximately 500bp on either side of mutation, without...

Day 1: Plating the Mixed Population 1. Find microsatellite containing strains in the 80...

Bacterial Mutation Accumulation Experiments Background Mutation accumulation (MA) experiments involve periodically bottlenecking a population such that evolution...

Barrick Lab :: Previous Research Identifying Mutations that Promote Bacterial Evolvability Epistasis Poker. Early draws of cards that are more beneficial to...

Presentation List 2010 03 17 MSU GEDD Next Gen.pdf: 2010 03 17 MSU GEDD Next Gen.pdf

Postdoctoral Fellow Position in the Barrick Lab Earliest Start Date: June 2011 Status: OPEN Posted March, 16, 2011 A position is open for a postdoctoral...

Barrick Lab :: News Archive August 2014: Graduate student Michael Hammerling is awarded a UT Graduate School Named Continuing Fellowship. April 2014: Graduate...

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Michael Hammerling Michael Hammerling MichaelHammerling 22 Nov 2011

Megaprimer whole plasmid cloning aka MEGAWHOP cloning aka Overlap Extension PCR cloning We describe two approaches to MEGAWHOP in this protocol page. Approach...