Eliminating the Kanamycin Resistance Cassette from Keio Strains Day 1 1 Make electrocompetent cells of the desired strain. 1 Transform with pCP20. This plasmid ...

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 ...

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 ...

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 ...

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 ...

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 ...

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

Competition Assays for Evolvability Lines Serial transfer of 3.5 µl of culture to 900 µl of fresh medium achieves 8.0120 generations per day (assuming the 3.5 µl is ...

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 ...

Gene Gorging Neutral Markers This procedure has been used to create defined Ara revertants of Escherichia coli B (REL606) and Mal derivatives of Escherichia coli ...

Gene Gorging Neutral Markers This procedure has been used to create defined Ara revertants of Escherichia coli B (REL606) and Mal derivatives of Escherichia coli ...

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 ...

Genomic DNA Preparation for Whole Genome Re sequencing of E. coli Current RTSF Instrument Information 454 Pyrosequencing (GSFlex machine) Read length is 100 ...

Label Templates Templates for printing labels...

Competition Assays for the Lenski Long Term E. coli Lines Day 2 Revive the strains to be tested from the freezer by growing them in one flask or test tube each ...

Making Electrocompetent E. coli Cells This procedure makes enough electrocompetent cells for 2 3 transformations. 1 Grow an overnight culture of each strain ...

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

Liquid Media LB Synonyms: TBYE 10 g Tryptone 5 g Yeast Extract 5 g NaCl Add dH2O to 1 liter. Autoclave. Luria Bertani Note: This is different ...

Phage Lysate Preparation Optimized for the production of high titer T4 lysates (1010 1012 PFU/ml). It is also suitable for phages T5 and T6 but tends to give lower ...

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 ...

Primer Design General guidelines 1 The lengths of primers should be 17 25 bases. 1 GC% should be 40 60% 1 Melting temperature (Tm) should be approximately ...

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

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

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 ...

Standard PCR Reaction 10 primer 10x dNTPs 10x Buffer 100x taq Whole Cell PCR Grow fresh overnight cultures of the cells to be used as template in LB ...

Primer Design Primers sequences and sometimes stocks already exist for some genes: topA PCR reaction Check PCR and Quantify DNA Yields Run 2 5 µl of sample 2 ...

Making Electrocompetent E. coli Cells Stratagene UV Crosslinker (located in the Schmidt lab). Prewarm the machine by running it for 2 minutes. Then set the machine ...

Tetrazolium marker for follow up competitions ASKA(AG1) is Ara–, Mal– Still need a marker... gene gorge deletion in ribose gene from Keio? Keio(BW25113 ...

Cloning and Strain Construction Projects Strain library evolution experiments KanR and empty PCA24N plasmids as ASKA controls Mal version of AG1 to follow ...

nop infB strain construction 1 REL606 with infB1 allele from JEB154. 1 JEB154 with ancestral REL606 allele. primer name sequence RL1041 ...

CamR Keio control strain construction General approach: use the Datsenko and Wanner gene replacement protocol (which is how the Keio strains were constructed) to add ...

Move Kishony cfp/yfp into REL606 background P1 transduction fails, probably due to the restriction modification system of the E. coli B derived REL606 differing ...

manB cpsG deletion 1 Amplify negative and positive selection cassette encoding sacB and cat from pKO3 adding genome specific regions to each end. Try two different ...

Protocols and Laboratory Reference Organization Label Templates Sequence Analysis Primer Design PCR Gene and Genomic Sequencing Bacterial ...

Publications Barrick JEPublication Network Graph by 26. Barrick, J.E. , Yu, D.S., Yoon, S.H., Jeong, H, Oh, T.K., Schneider, D., Lenski, R.E., and Kim, J.F. 2009 ...