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Back to Golden Gate Protocols Part Plasmid Assembly Once you have designed your part and either amplified with PCR or ordered the desired gBlock (as described here...

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

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...

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

Back to Golden Gate Protocols First Stage Plasmid (Transcriptional Unit) Assembly Once you have all your desired part plasmids built you can assemble them into Transcriptional...

Back to Golden Gate Protocols Second Stage Assembly Second Stage assemblies are used to build plasmids out of multiple transcriptional units (Protocol found here...

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...

Aphid Care and Protocols Rearing and Caring for Aphid Species Protocols and Primers

Media amendments Preparation: Generally, prepare 30 50 mL of solution in a 50 mL conical tube. Then, filter sterilize solutions by pushing them through a 50 mL syringe...

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...

`In plate` / solid medium transformation of Acinetobacter baylyi ADP1 The following protocol is for single colony `in situ` (in plate) transformation with minimal...

Acinetobacter baylyi ADP1 Genome Manipulations by Overlap Extension PCR Overlap PCR Construct Generation The following is a standard procedure designing and constructing...

Acinetobacter baylyi ADP1 Golden Transformation Overview The following protocol is to be used as a substitute for overlap extension PCR for constructing double stranded...

Acinetobacter baylyi ADP1 Genome Manipulations Genome manipulations in Acinetobacter baylyi ADP1 can be performed without the need for exogenous recombinase expression...

Acinetobacter baylyi ADP1 Overview ADP1 Resources Genome Sequences type strain (GenBank Format) In general, use this genome as a reference when referring...

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

16S rRNA Sequencing to Identify Unknown Microbes Ever wonder what that contaminant in your culture is? Need to accurately identify an environmental isolate? Overview...

Protocol Updates Needed this page on DNA sequencing needs to be updated and be made more for the general public than to the lab General guide for sorting...

Phage Lysate Preparation We have used this protocol with phages T4, T5, T6 and T7. The general procedure should work with most lytic E. coli phages (although lysis...

Barrick Lab :: Laboratory Protocols Updating Protocols How to Create a Protocol Best practices for designing a protocol and for putting it on the lab Wiki....

How to Create a Protocol Tips for Design You should generally organize a protocol to have sections that are relevant from this list: Supplies (materials, strains...

Calculating Growth Rates with Grofit The following are instructions for calculating growth rates using Grofit, an R package that is no longer supported by the current...

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...

Gel Electrophoresis Overview Gel electrophoresis separates DNA fragments based on size. Electric current moves the negatively charged DNA through the gel, which slows...

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...

Sequencing/Genotyping Primer Design This protocol is specifically for designing primers to PCR amplify a target region of interest from a genome and re sequencing...

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...

Major version notes Runs prior to February 2020 had a variety of formats and sample submission requirements. Suggested that individual runs be consulted on utbox for...

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...

Co culture Competition Assays The instructions below include the basic protocol. Be sure to check whether there are variations needed for your specific samples! For...

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...

Gene Gorging Evolved Alleles This procedure can be used to directly create unmarked mutations in the E. coli genome. Transform Gene Gorging and Donor Plasmid...

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 ...

Evolution Experiments Introduction An evolution experiment typically consists of evolving one or more ancestor strains in laboratory culture and assessing the types...

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