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Calendar
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
May 29
Roxanne
- prepared glycerol stocks for autoclaving
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
July
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.
August
September
October
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