Berk2010-Tahoura

Tahoura Samad 13:46, 8 June 2010 (EDT)

PDD Subgroup Work

• all five transformations grew up well. The 2343 and 2344 appeared red.
• Picked colonies for 2343(spec), 2344(spec), 1256-1934(spec), pb6(Cam) and MG1655 (LB) in a 24 well plate. Put in 37 C shaker. Check on them at 4 pm.

Manual Assembly of iGEM10_13 and iGEM10_20

• began manual assembly of assembly tree for iGEM10_13 and iGEM_20. (create iGEM10_008, 009, 014, 015, 016.)
• set up Lefty and Righty digests and incubated: incubation should be done at 2:50 pm
  • Digested 8+4, 7+11 (x2), 9+20 (x2) with Lefty MasterMix.
  • Digested 9+3, 8+1, 7+2, 7+19, 8+12 with Righty MasterMix.
• Zymoed the 8 digests.
• Set up ligation reactions;
• Ligate
  • Followed changed protocol (see 6/7/2010):
  • Set up 5 ligations (made MasterMix recipe x6)

Mastermix
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
0.5uL T4 DNA Ligase
DNA
3uL Lefty vector
3uL Right vector
Start time (4:15 pm, End time 4:45 pm)'

• transformed ligation into JTKO49 (check name with Daniela) (from -80 freezer) because there were no lefty and righty cells left.
• plated and put in incubator
  • iGEM10_016, 008 on CK
  • iGEM10_015, 009 on AC
  • iGEM10_014 on KA

Manual Assembly of iGEM10_001, 002, 003

• Ran gel of colony PCR
• Parts looked correct.

To Do

• create glycerol stock of pb6 for -80 freezer. Cross streak to check for contamination
• transform the colonies we picked (2343(spec), 2344(spec), 1256-1934(spec), pb6(Cam) and MG1655 (LB) in a 24 well plate. )

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

PDD Subgroup Work

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

Manual Assembly of iGEM10_003

• Digestion
  • prepared Righty Master Mix (see group lab notebook)

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Lefty Mastermix Recipe (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 added to 7+23, 8+6, 7+5
• L mastermix added to 9+8, 7+11, 9+20

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• 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

• Incubated 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.

Additional Notes

• Zymo can be used to of get rid of restriction enzymes. In this case, we can't heat kill because BamH1 survives.

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
• Bring folder for iGEM assembly trees.
• coat hangers?