Team:Lethbridge/Notebook

== Calendar ==

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May

May 5

Roxanne, Megan, Alix, Mackenzie, Sebastian

• prepared Lb Media in test tubes -prepared 250mL LB agar for plates

• Prepared 500nM Tris-HCl (pH 8.0), EDTA (pH 8.0, 500nm) TE Buffer (pH 8.0), GTE (Solution I), 2M NaOH, 10% SDS

May 6

Roxanne

• Autoclaved LB test tubes, LB agar, Tris-HCl, EDTA, TE, GTE

• Added ampicillin to LB agar (100ug/mL) -Poured LB + amp plates

May 12

Roxanne

• Prepared LB agar
• added Tetracyclin (100ug/mL) –
• Poured LB + ampt plates

Roxanne, Alix, Ashley, Megan, Mackenzie, Kirsten

• transferred pE43 (DH5α) cells from gylcerol stock to 5mL LB+Tet test tube (x8)
• placed in orbital shaker overnight at 37°C

May 13

Roxanne

• Removed test tubes from shaker (16hrs incubation) and placed in deli fridge

May 14

Roxanne, Alix, Ashley, Mackenzie, Kirsten

• Preformed alkaline lysis miniprep on pE43 plasmid -lost plasmid from 2 tubes

• obtained 6X pE43 plasmid (2X ohbR, 2x ohbB, ohbA, ohbC)

May 19

Roxanne, Alix, Ashley, Kirsten, Megan, Mackenzie

• Gel Electrophoresis : 1% agarose in TAE

Lane 1: Ladder

Lane 2: ohbR1

Lane 3: ohbR2

Lane 4: ohbC

Lane 5: ohbB1

Lane 6: ohbB2

Lane 7:ohbA

Lane 8:ohbC

Lane 9: ohbB1

Lane 10:ohbB2

• DNA quantification by UV spec 260nm : 2.013 280nm: 1.105 260/280 : 1.984 Concentration: 2517µg/mL PCR of ohbA, (ohbB)x2, ohbC, (ohbR)x2

• Took picture of the gel - it did not turn out.

May 21

Roxanne

• Made 50X TAE Buffer
• Running PCR amplicons & RS/pSB1A2 ligation product on a 1% agarose gel
• Jeff gave tutorial on primer design and quick-change mutagenesis to Kirsten, Ashley, Mackenzie, Fan, Lisza, Roxanne

May 26

Roxanne, Alix, Megan, Mackenzie, Kirsten

• Gel did not turn out gave tutorial on open wetware and the igem registry

May 28

Roxanne

• reran the gel from may 19th, only primer dimers present

Roxanne, Ashley, Alix, Kirsten, Mackenzie, Megan

• Redoing the PCR of ohbA, ohbB, ohbC and OhbR with Phusion DNA Polymerase

Roxanne

• refilled the tip boxes

May 29

Roxanne

• prepared glycerol stocks for autoclaving

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June

June 1

Roxanne

• Ran a 1% agarose in TAE gel of the PCR products from May 28th.
• 5µL of ladder, 2µL of DNA

Lane 1: ladder

Lane 2: ohbA (conc.1x)

Lane 3: ohbA (conc.1/10)

Lane 4: ohbB(1x)

Lane 5: ohbB(1/10)

Lane 6: ohbC(1x)

Lane 7: ohbC(1/10)

Lane 8: ohbR (1x)

Lane 9: ohbR(1/10)

Lane 10: ohbR(1x)

• Gel did not show any amplification

Mackenzie & Roxanne

• Transformation of pSB1A3 into DH5α

• Made 2 aliquots of pSB1A3 and 1 of pUC19

• plated pSB1A3-1: straight and dilute, pSB1A3-2: straight and dilute, pUC19: straight only incubated overnight at 37°C

June 3

Roxanne

• positive control worked
• Think the transformation worked but slight problem. The plasmid contains a suicide gene. Therefore, any cells that took up the plasmid would die.
• planning new genes to transform
• GENE LOCATION pSB1A3 + IPTG Inducible RFP 1-1k Bba_I13522, pTET GFP 2-8A Bba_I13521, pTet mRFP 2-6O Bba_I13600, pTET CFP 1-24E BBA-B0015, double terminator 1-23L

June 4

• Picked some glycerol stock GFP for the AIF visit

• made ampicillin plates 371.39g/mol X100mmol/LX0.002L = 74.278mg

June 15

Transformed the pTet and EYFP genes from the Biobrick registry

June 16

• Picked colonies in the early morning for Mini prep in the afternoon

• Restriction digested the plasmids: pTet with SpeI and PstI, EYFP with XbaI and PstI

June 17

• Quenched the restriction digests from the day before

June 18

• Checked the concentrations of:
  • Riboswitch-1 : 4549u/.mL
  • Riboswitch-2 : 4283ug/mL
  • rpsA TIR-1 : 106ug/mL
  • rpsA TIR-2 : 90ug/mL
  • rpsA TIR-3 : 78ug/mL
  • rpsA TIR-4 : 74ug/mL
  • rpsA TIR-4b : 67ug/mL
  • rpsA TIR-5 : 70ug/mL

in order to send for sequencing with the UR and UF2 primers

June 22

• Picked one colony each from DH5a + pTet and DH5a + EYFP.
• Grown overnight at 37 degrees in LB +100ug/mL Amp (5mL culture tubes)
• From mini-preps of EYFP(1&2) and pTet (1&2) did preparative restriction digests
  • pTet with PstI and SpeI
  • EYFP with XbaI and PstI
• In 37 degree water bath for 1hr and 20 min. Quenched at 60 degrees for 15 minutes.

June 23

• Ran a 1% agarose gel (1mL 50X TAE with 49mL Milli-Q H2O and 0.5g of agarose).

Lane 1: 1kb ladder

Lane 2:EYFP1A

Lane 3: EYFP1B

Lane 4: EYFP1B runover

Lane 5: EYFP2A

Lane 6: EYFP2B

Lane 7: pTet-1A

Lane 8: pTet-1B

Lane 9: pTet2-A

Lane 10: pTet-2B

• Samples were 2uL of dye and 10uL of DNA.
• The gel was unsuccessful. Possible reasons include: too short of an incubation during restriction digest or the enzymes were not functioning properly

June 24

• Mini-Preps of pTet and EYFP
  • 750uL of culture from the fridge into microcentrifuge tubes. Pellet at 13000rpm for 2 min. Removed supernatant and added the rest of the culture. Repelleted at 13000rpm for 2 min.
  • followed protocol according to Qiagen mini-prep protocol on page 2
• Restriction Digest of EYFP and pTet
  • EYFP restricted with XbaI and PstI
  • pTet restricted with PstI and SpeI
    • Double digest:1uL buffer tango, 8uL DNA, 0.5uL of each enzyme
    • Single digest (1 for each enzyme): 0.5uL water, 1uLbuffer tango, 8uL DNA, 0.5uL enzyme
    • Negative control: 1uL water, 1uL buffer, 8uL DNA
    • 8 tubes total in to the thermocycler for 8hrs at 37, 15min at 60 then held at 4 before going into the -20 fridge.

June 25

• Ran a gel of the digested EYFP and pTet and controls from the previous day.
• Used 0.3g of agarose in 30mL of 1X TAE
• made stock solution of 1x TAE
• Ran gel at 100V for 1hr

Lane 1: 1kb ladder

Lane 2: Control-no enzyme(pTet)

Lane 3: pTet-pstI

Lane 4: pTet-SpeI

Lane 5: pTet-SpeI/PstI

Lane 6:EYFP-no enzyme

Lane 7: EYFP-PstI

Lane 8: EYFP-XbaI

Lane 9: EYFP-XbaI/PstI

Lane 10: 1kb ladder

• Gel melted
• Made 2 pTet and 2EYFP 5mL culture tubes with LB and Amp(100mg/mL). Left in incubator overnight at 37°C

June 26

• Cells harvested from pTet and EYFP cultures into 4 tubes. Spun down and LB supernatant removed. Stored in -20 for future use.

June 30

• 200mL cultures of cells with pTet, EYFP and CFP grown in LB and 100ug/mL Amp harvested by centrifugation then maxi-prepped.
• Maxipreps
  • incubated cell pellets in 3mL of ALSI and incubated at RT for 15min
  • 6mL of ASL2 added and incubated at RT for 10min
  • 4.5mL of AlS3 added and placed on ice for 10min
  • Spun at 5000g at 4 for 15min
  • 5mL of each phenol and chloroform added to tubes containing resulting supernatant
  • spun for 10min at 4 and 5000g, upper aqueous layer saved
  • 5mL of chloroform added to aqueous layer and spun for 10 min at 4 and 5000g. Supernatant saved
  • 0.6 volumes isopropanol added to each tube and placed in -20 freezer for 30min –
  • spun for 10min at 4 and 5000g
  • pellet wasted with EtOH and airdried overnight

• 5 culture (500mL) flasks of LB made: 10g tryptone, 5g yeast extract, 10g salt and 1L water

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July

July 2

• DNA samples dissolved in 1000uL water, 100uL of RNAse added. Made 1/10, 1/100 and 1/1000 dilutions of pTet, CFP and EYFP.

July 3

• Ran a 1% agarose gel at 120V for 60 minutes.
• 2uL DNA samples with 2uL 6x loading dye

Lane 2: 10uL of 2kb ladder,

Lane 4:undiluted pTet

Lane 5: undiluted CFP

Lane 6:undiluted EYFP

Lane 8: 1/10pTet

Lane 9: 1/10 CFP

Lane 10: 1/10 EYFP

Lane 12: 1/100 pTet

Lane 13: 1/100 CFP

Lane 14: 1/100 EYFP

Lane 16: 1/1000pTet

Lane 17: 1/1000 CFP

Lane 18: 1/1000 EYFP

July 6th

Jeff, Mackenzie, Ashley

Using 1/10 and 1/100 dilutions of pTET and EYFP to perform large scale digests.

• pTET:PstI and SpeI
• EYFP:XbaI and PstI

4 reactions each:

• single digest (PstI/SpeI and XbaI/PstI)
• double digest
• no digest

100µL each, means we need 400µL total for each.

1/10 dilution=40µL DNA, 360µL water

1/100 dilution=4µL DNA, 396µL water

Reaction set up:

• 88µL DNA
• 10µL buffer Tango (10x)
• 1µL RE1 (or water)
• 1µL RE2 (or water)
• =100 µL total

16 reactions run overnight @ 37° C

Kirsten, Lisza

TetR Q04400-plate 1 well 16p-pSB2K3

LacI promoter-R0010-plate 1 well 1d-Amp

pBAD-I13453-plate 1, well 1n-pSB1A2

strong RBS-B0030-plate 1 well 1h-pSB1A2

med RBS-B0030-plate 1 well 2I-pSB1A2

10µL water into wells

Transformation:

2µL DNA (TetR, LacI, pBAD, med RBS, strong RBS) into 25µL competent DH5α cells (from the -80 fridge). Pipet up then down and swirl. One ice for 30min, then heat shock at 42° C for 45 sec. Ice for 1 min, add 250µL LB broth (from fridge) and incubate in shaker for 1 hour. Plate onto ampicillin plates (pBAD, LacI, strond and med RBS) and kanamycin plate (TetR) into incubator overnight. Control: 2.5µL water and 2.5µL DH5α.

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August

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September

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October

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