Berk2010-Tahoura

Tahoura Samad 14:15, 7 June 2010 (EDT)

referenced Jin's notes:

• (with Conor) Transformed Mg1655 cells (from -80 freezer) from Jin with two PDD 2343, 2344( from freezer box).
  • Diluted DNA by 10x ( 9 μL dH20, 1 DNA), added 1 μL to cells.
    
• pB6 (BAC) see image, arrived from Germany. Wear gloves because its BSL 2. Currently in fridge. TIm streaked it on a plate. (Note: Streak such that the concentration is reduced as you go along.) The part we need arrived in bacteria, so we need to grow them up and miniprep them to get the DNA we need.
  
• Transformed MG1655 cells with pBca1256-Bjh1934 (from Jin's Overflow box 1). Tube name: 1256-1934 into MG1655
  
• Plated our transformations (2343 in MG1655 and 2344 in 1655) on Spec plates (yellow). Plate names: 2343 into MG1655, 2344 into MG1655
  
• Created spreadsheets for iGEM Freezer boxes
• Tried to enter part information into Clotho, but were unable to save in FlatData
• entered parts into Google Group spreadsheet
• plated transformation of MG1655 with pBca1256-Bjh1934. Plate name: 1256-1934 into MG1655
• prepared Righty Master Mix (see group lab notebook)
• Helped transposase group with Manual Assembly of iGEM10_003
  • Digestion

L mastermix added to 9+8, 7+11, 9+20 Lefty Mastermix Recipe Lefty MasterMix (for 4uL miniprep DNA) 4uL water 1uL of NEB2 0.5uL XhoI 0.5uL BamH1 Righty MasterMix (for 4uL miniprep DNA) 4uL water 1uL of NEB2 0.5uL XhoI 0.5uL BglII R masterimix (see protocols) added to 7+23, 8+6, 7+5

• Ligation:

Normal protocol with slight changes:

• • Mastermix Recipe
    • 6.5uL ddH2O
    • 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
    • 0.5uL T4 DNA Ligase

DNA Recipe

• • • 3uL Lefty vector
    • 3uL Right vector

Incubate on bench until 5:37pm. Does not need to sit for full 10 minutes on ice. Tim says one or two minutes should be fine. Heat shock for 90s. 

• Transformed 9+20,7+5 (iGEM10_003) and 9+8, 7+23 (iGEM10_001) into the Righty strain. And 7+11, 8+6 (iGEM10_002)into the Lefty strain. Gave to Tim to plate.

• Note: Zymo

Means of getting rid of restriction enzymes. Can't heat kill because BamH1 survives. Eluted with 10uL of H2O. Products tube names: 7+11 L, 9+8 L, 9+20 L.

To Do

• Remind Tim to streak more of the MG1655 DONE.
• Remember to pick the MG1655 colonies tomorrow.
• Pick and miniprep BAC from Germany
• Pick and miniprep 0343, 0344, pBca1256-Bjh1934