Berk2010-Tahoura

Tahoura Samad 16:16, 15 June 2010 (EDT)

PDD Subgroup Work

• ALERT!: Mg1655 competent cells are probably bad. CHECK WITH TIM
• made glycerol stock of BAC with pBca1256-Bjh1934. See protocol online. Scanned on SimpleScan and logged in -80 stock on iGEM parts and data google doc.

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• Started competent cells for BAC WITH pBca1256-Bjh1934 (Medium Scale)

1. added 500μL of overnight-grown culture to 50mL Spec/Cam LB in an Erlenmeyer flask

• put flask in shaker at 11:05 am. Check at 1:05 pm.
• cells were not really ready at 1:05, kept in incubator until 2:05 pm.

2. checked overnight culture at 2:05 by spinning down 100 μL. Saw a pretty small white pellet.
3. Transfered culture to a 50 mL falcon tube that was sitting on ice.
4. Chilled overnight culture for 15 minutes to stop growth.
5. Got chilled rotor from cold room. (was in there for a couple of hours.) Changed rotor and rotor program.
6. Spun down cells at 4100 rpm for 13 minutes at 4 C.
7. Poured of supernatant and resuspended in 2.5 mL of ice cold TSS.
8. Made 25 98 μL aliquots in 0.5 mL eppendorfs on dry ice and put them in the same rack as our other competent cells in iGEM competent cell box 2. the cells are labeled "5" and have been entered into the iGEM competent cell google doc.
9. Streaked for contamination on Amp, Kan and Cam/Spec. Should grow on Cam/Spec, but not Amp, Kan.
Plate names: iGEM control for competent cells BAC with 1934

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• Did genomic miniprep of Pb6.

Same steps as regular miniprep with the following changes.
This procedure is identical to the normal miniprep procedure except the step in red, and less culture is prepped
1. Pellet 1.5 mL saturated culture by spinning full speed, 30 seconds.
2. Add 250uL of P1 buffer into each tube. Resuspend the cells using
a vortexer.
3. Add 250uL of 2% SDS (stored in a 50 mL conicol)
(actually stored in falcon tube. Also, this step replaces the P2 step.
4. Incubate at 55 degrees until clear (roughly 15-20min?).
5. Continue as usual with N3 step.

• stored in iGEM working box. tube name: g. pB6
• added 250 μL of 2% SDS after adding p1. Incubated at 55 until clear. check at 12:00 pm

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• ALERT checked on transformations of MG1655 with pBca1601-Bjh2331,2333,2348,2349 and MC1061 with pBca1601-Bjh2331,2333,2348,2349.
• MC1061 grew up really well, tons of really small colonies.
• MG1655 had few, big colonies.

1655-2331 (50-100 colonies)
1655-2333-(35 colonies)
1655-2348 (35-50 colonies)
1655-2349(35-50 colonies)

• Picked two colonies of each (16 total) and am growing them up in a 24 well block in the 37 C shaker.

Additional Notes

• iGEM competent cell box 2 is in the same rack as iGEM competent cell box 1, in the back column.

To Do

• Important Check with Tim about the competent MG1655 cells.
• check controls for competent cells for BAC with pBca1256-Bjh1934 in MG1655. Kan and Amp should be negative and Cam/Spec should be positive.
• check on transformations of pBca1256-Bjh2343 with pB6 (1:10 1 μL, 1μL, 3μL). Pick and grow up for competent cells and glycerol stock if there are any.
• ask Tim what we should do with the MC1061 and MG1655 with pBca1256-Bjh2331,2333,2348,2349 that are growing up in the 37 C shaker.
• streak out ligation Ecoli.

Tahoura Samad 14:53, 14 June 2010 (EDT)

PDD Subgroup Work

• Tim says the 10 μL BAC with pBca1256-Bjh2344 had two colonies, so he picked one, grew it up and made glycerol stock in the minus 80 for us.
• picked pB6 so we can do a genomic miniprep on it and try to create BAC with pBca1256-Bjh2343 again.
• picked BAC with pBca1256-Bjh1934 3μL so we can create a glycerol stock and competent cells.
• put both in shaker upstairs to incubate overnight. start time: 11:00 pm
• restarted Eco/Bam transfer of pBca1601-Bjh2331, 2333, 2348, 2349 into Shelley's AK 1601 because we forgot to add magnesium when we started the cloning steps on Friday.
  • To make I M magnesium sulfate

-Added 12.4 grams of Magnesium sulfate to 50 ml of water in a falcon tube. Screwed on filter and vacuum filtered.

• incubated at room temperature for 30 minutes: start time: 1:45
• transformed Eco/Bam transfer for all 4 parts into MG1655 (4) and MC1061 cells. The MC1061s are in the -80 freezer, same row as usual, farthest left rack, second row, bottom box. They have a blue stripe. started transformation at 3:20 should be done at 4:20added 10μL of MgS04.
• plated 400 μL of MgSo4 onto each of the Kan, Amp plates. Then plated 70 μL of each of the transformations. Set in 37 incubator.

New project to get ColE2 and pir site into a strain of TIm's bacteria so we can combine the transformation and ligation step

• get strain out of freezer. Its in row B8. In -80 freezer, 2nd row from the top leftmost rack, in blue bottomed box.

To Do

• check on pBca1256-Bkh1934 and pB6. Make glycerol stock and competent cells of pBca1256-Bjh1934 and genomic miniprep pB6.

Tahoura Samad 13:12, 11 June 2010 (EDT)

PDD Subgroup Work

PbCA1256-bJH2344 with 1 μL
PbCA1256-bJH2344 with 3 μL
PbCA1256-bJH2343 with 3 μL
PbCA1256-bJH2343 with 1 μL

• checked on second round of BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344, and BAC with pBca1256-Bjh1934 plates tomorrow. No growth for BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344 (1 μL and 3 μL) but ~50 colonies on pBca1256-Bjh1934: 1 μL and decent growth for pc.
• Talked to Tim. Tim says since BAC is 40 kb, not 15 kb, the miniprep we used is not really good, so I'll re-miniprep BAC on Monday.
• In the mean time, I retransformed BAC with pBca1256-Bjh2343, and BAC with pBca1256-Bjh2344 and added 10 μL of undiluted pB6 miniprep.Incubation start time 11:04pm
• plated entire transformation on Cam Spec plates.
• Eco/Bam transfered 1601CA Bjh 2333 L, 1601CA Bjh2348 L, 1601CA Bjh2349, and 1601CA Bjh 2331 in pBca1601 AK. We did this because BAC is CAM resistant, so if both parts are Cam resistant, it is possible to lose BAC, which is only single, and not be able to tell. So in the end, we will end up quadruple plating our stuff.

Creating Competent Lefty, Righty Cells, (MC1061 pir 1)

• created glycerol stock for MC1061 pir1 lefty and righty cells. Logged on google doc and put into -80 freezer
• helped Tim a little to make competent lefty righty cells.

Tahoura Samad 16:56, 9 June 2010 (EDT)

PDD Subgroup Work

• checked controls for competent cells. They looked good with no growth on Amp, Cam and Kan and growth on Spec.

Negative Control for Amp
Negative Control for Kan
Negative Control for Cam
Positive Control for Spec

• checked on transformations with BAC. All failed. 0 colonies for both plates. Possible reasons for failure: We added twice as much KCM as we should have. Also, Tim said some BACs are just hard to transform.

Transformation of pBca1256-Bjh2343 in MG1655 with BAC
Transformation of pBca1256-Bjh2344 in MG1655 with BAC (left) ;Transformation of pBca1256-Bjh1934 in MG1655 with BAC (right)

• threw plates away in red garbage
• redid transformations with 1μL, and 3 μL of undiluted pB6 miniprep. Scaled down KCM to 15μL because our cell aliquots are only 98 μL Start time in incubator: 11:08 am.
• plated all 159ish μL of each transformation on Spec, Cam plates (blue yellow). plate names: 1 μL BAC with pBca1256-Bjh2343. 3 μL BAC with pBca1256-Bjh2343. 1 μL BAC with pBca1256-Bjh2344 3 μL BAC with pBca1256-Bjh2344, 1 μL BAC with pBca1256-Bjh1934, 3 μL BAC with pBca1256-Bjh1934.

Assembly of iGEM parts 001 and 009

• started miniprep of assembly 2 with Amy
• cut out bands from Amy's gel. Lane 1: 1L (desired length: about 1100) Lane 2: 1R (~1300) Lane 3: 9L (~1200) Lane 4: 9R (~1500)
• the point of the gel purification was to get rid of parent vector and to select for the side of the vector we want,
• the part lengths were obtained from the part calculator sequences and the lengths of the digested pieces that would ultimately be included in the iGEM10_001 or 009 composite part after ligation and appropriate antibiotics selection.

I combined the 1L with the 1R and the 9L with the 9R gel fragments and performed a Zymo Gel Purification, eluting with 17uL of water for each.

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• Ligation:
• Set up ligation mastermix

Mastermix
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (black-striped tubes)
0.5uL T4 DNA Ligase

DNA 6uL of L+R gel purified DNA
Added DNA to MM at 3:54pm. The ligation will be done at 4:30pm.

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Assembly with Gibson Oligos

• Conor showed me how to design Gibson Oligos using the Gibson Oligo Spreadsheet
• Designed the Gibson Oligos for iGEM10_20 and checked Conor's oligos for iGEM10_13.
  • Note: 20 mers can be longer than 20 so that Tm is at least 55. Additionally, oligos need to end on a g or a c.

To Do

• check on BAC with pBca1256-Bjh2343, BAC with pBca1256-Bjh2344, and BAC with pBca1256-Bjh1934 plates tomorrow.
• pick colonies and grow up to make BAC -80 freezer stock

PDD Subgroup Work

• Created -80 Stocks for pBca1256-Bjh1934 in MG1655, pB6 in DH5-alpha, MG1655, Bjh2343 in MG1655, Bjh2344 in MG1655r using this protocol
• scanned and logged these stocks as the first iGEM -80 stocks (created a stock box for -80). Scanner is located next to pipetting robot. Spreadsheet for -80 stock is located on 140L parts spreadsheet on google docs.

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• Did Miniprep of PB6 starting with 2mL of the overnight culture. Stored eluent (50 μL) of DNA in iGEM working box.

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• Made Competent Cells for pBca1256-Bjh1934 in MG1655,Bjh2343 in MG1655 and Bjh2344 in MG1655:

Recipe
1. added 500μL of overnight-grown culture to 50mL Spec LB in an Erlenmeyer flask for MG1655: (1:100)
2. added 1mL of overnight-grown culture to 50mL LB (no AB) in an Erlenmeyer flask. For this one, we doubled the amount of cell culture because we added glycerol to our cells, so they were half the dilution they should have been.
3. Put both flasks put on shaker in lab at 12:00
4. Took cells out of shaker. Had to put the MG1655 back in for longer because they grew very poorly. Probably because we added glycerol.
5. Followed protocol for large scale competent cells with the following amendments:

• spun cells down for 13 minutes at 4100 RPM. MAKE SURE TO BALANCE THE CENTRIFUGE to within 1 gram.!!!!

6. While centrifuge was running, we got .5 ml eppendorfs and labeled them 1, 2, 3, 4, while keeping them on dry ice. Dry ice is so the cells flash freeze, which is good if you are working slowly. The numbers 1, 2, 3, 4 correspond to different competent cells, and the key can be found in the iGEM parts and data spreadsheet on google docs.
7. Removed falcon tubes from centrifuge and poured off supernatant into a bottle in the sink. Add bleach etc.
8. Resuspended pellet by vortexing in 2.5 mL of TSS, which can be found in the plate fridege, top right back.
9. Put on ice, and aliquoted 98 μL in 25 Eppendorfs for each of the four.
10. Put in -80 freezer. Third shelf? (or same shelf as JTK049, rack furthest to the rights.
11. Set up positive and negative controls for our competent cells

• Streaked cells on Cam, Amp, Kan and Spec. Cam, Amp, and Kan should come up negative for 1, 2, 3. Cam, Amp, Kan and Spec should for 4.
  

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• Transformed BAC into 1, 2, 3.
• Plated on Cam, Spec plates.

To Do

• check controls for competent cells. Kan, Cam and Amp should be negative (no cells) spec should be positive. Exception: 4 (MG1655) should be negative for all 4 plates. DONE
• make competent cells for pBca1256-Bjh2343 with BAC, pBca1256-Bjh2344 with BAC, pBca1256-Bjh1934 with Bac.
• Find out what BAC (PB6) is
• Look up what pBca1256-Bjh1934 is. (short description, part name)

Additional Notes

• shaker on fourth floor is not at exactly 37, closer to 33, 34, so things take longer
• make sure to scale down KCM by half for all the competent cells we made. (1, 2, 3, and 4)
• BAC stands for Bacterial Artificial Chromosome.

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.

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• Set up ligation reactions;
• Ligate
  • Followed changed protocol (see 6/7/2010):
  • (Amy)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)'

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• transformed ligation into Jtk049 (not methylated, 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.

Additional Notes

• Dishwashing room: on 4th floor. Can fit 15 blocks in a rack. Dishwasher on right is messed up.

To Do

• create glycerol stock of pb6 for -80 freezer. DONE
• Create -80 stock for all 5 and competent cells for everything but pB6.
• coat hangers? DONE

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. DONE
• Pick and miniprep BAC from Germany DONE
• Pick and miniprep 0343, 0344, pBca1256-Bjh1934 DONE
• Bring folder for iGEM assembly trees. DONE
• coat hangers?