User:Tk/Notebook/MF-xfm/2008/04/20

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] MF-xfm
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Transformation results

 * 73 transformants from last week's transformations
 * Most from transformations with low cell density
 * Most from transformations with 10 ul 1M sorbitol added prior to electroporation
 * nothing shows at 14 hours or 24 hours
 * Some colonies visible at 38 hours, more at 48, final at 60 or so

New standard transformation protocol

 * culture cells in 45 ml 1161 medium to light yellow/slightly turbid
 * Spin down and resuspend 3x with 45 ml EPB (no glycerol) on ice in chilled centrifuge
 * Resuspend in 5 ml EPB on ice
 * Aliquot 50 ul cells + 1 ul transposon DNA on ice
 * Add 10 ul 1 M sorbitol
 * Electroporate at 1.5 KV in 1 mm cuvette
 * Immediately add 850 ul 1161 medium, place on ice
 * Incubate 1 hour at 30C in the cuvette
 * Plate 300 ul on 15 ug/ml TET plates
 * Hold remainder in the cuvette for several days at 4C

Newly diluted transposase

 * Dilute 50 ul transposase (8 ug/ul) into 800 ul transposase storage buffer
 * Store at -20, label 500 ng/ul transposase

New transposomes

 * 20 ul transposon DNA (83 ng/ul)
 * 20 ul glycerol (use cut off tip for pipetting)
 * 40 ul diluted transposase
 * incubate 1 hour at 37C
 * hold at -20C

New transformations

 * standard protocol
 * use 10 ul water, 5 ul water, no addition, 5 ul, 10 ul, and 15 ul sorbitol (1 M) additions
 * Test for both positive and negative osmotic pressure changes, and narrower range than last time
 * Plate out all of sorbitol added versions (3 plates)
 * Plate out only 1 plate from water and no addition plates, save remainder at 4C
 * Plates labeled with -10, -5, 0, +5, +10, +15 for water and sorbitol additions (ul added)

Plans for genetics

 * Promoters
 * constitutive
 * heat shock
 * Reporters
 * YFP, mCherry
 * antibiotic
 * cat gene
 * recT recombination gene
 * Test constitutive and heat shock promoters with reporter
 * Test cat gene with constitutive promoter
 * Combine heat shock promoter with recT gene
 * Test recombination efficiency


 * }