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 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.
July 8
Jeff
Overnight cultures (200mL + antibiotics) of:
- PlacI (Amp)
- TetR (Kan)
- Med RBS (Amp)
Centrifuged at 5000g, 10 min, 4° for maxiprep
500µL each of each culture used to make glycerol stocks – duplicates made, stored in -80° freezer after flash freezing with liquid nitrogen.
EYFP insert from AGE separation (incubated in 600µL buffer QG overnight):
- +200µL isopropanol
- Incubated on a QiaQuick column
- 13000rpm, 1 min
Flow through replaced in original tube. 500µL QG applied to column, 1 min centrifugation. Colum washed w/ 750µL PE buffer (1 min centrifuge). Flow through discarded. 1 more min of centrifugation to get rid of buffer PE. DNA eluted w/ 25µL water (37°C for 10 min) also a second water elution w/ 25µL water (E2).
1/10 double digest of pTet heat incubated at 85°C for 10 min.
Maxipreps: 3mL ALSI added, cells responded. 500µL lysozyme added. 15 min @RT, 6mL ALSII added. 10 min at RT. 4.5mL cold ALSIII added, 10 min on ice. Left a 4°C
July 13
Setting up ligations:
1µL ligase (stock)
10x T4 DNA ligase buffer
1/10 double digested pTet est 20ng/µL
E1 (60% yield) = ~5ng/µL; ~1/3 insert size/pTet
Control needed:
- 1/10 double digested pTet, no ligase
- 1/10 double digested pTet + ligase
- 1/10 double digested pTet + 3x vol of insert
- 1/10 double digested pTet +6x vol of insert
Reaction 1:
- 1µL T4 DNA ligase 10x buffer
- 1µL double digest pTet
- 8µL water
Reaction 2:
- 1µL T4 DNA ligase 10x buffer
- 1µL double digest pTet
- 0.5 µL T4 ligase
- 7.5µL water
Reaction 3:
- 1µL T4 DNA ligase 10x buffer
- 1µL double digest pTet
- 0.5 µL T4 ligase
- 3µL E1 EYFP insert
- 4.5µL water
Reaction 4:
- 1µL T4 DNA ligase 10x buffer
- 1µL double digest pTet
- 0.5 µL T4 ligase
- 6 µL E1 EYFP insert
- 1.5µL water
Incubated at 37°C overnight
Kirsten, Mackenzie
Chloroform extraction of medRBS, pLacI, TetR, that Jeff started.
pBab & strong RBS cloudy and phenol chloro done twice.
July 14
Megan, Mackenzie, Alix
Transformations of ligation reactions
Took 2µL of DNA from 1, 2, 3, 4 and added to 25 µL of DH5α cells. Incubated on ice for 30 min. Heat shocked at 42°C for 45 sec. Back to ice for 1 min. For ligations add 500µL of LB broth to each tube, working sterile using a Bunsen burner. Incubate in shaker at 37°C for1 hour at 200 rpm. Made up two plates (Amp) of each, 100µL and 400µL.
July 16th
Ashley, Mackenzie, Roxanne
Pelleted pTet-EYFP ligation reactions 3 and 4 using at 13000rpm for 1 min.
Mini-prep of pTet-EYFP ligation reactions 3 and 4 using Qiagen kit + protocol.
July 20th
Lisza
Our genes: added miiliQ water
- 10µL to N-term Arg tail, concentration = 200ng/µL
- 10µL to c-term Arg tail, concentration= 200ng/µL
- 20 µL to lumazine, concentration= 250ng/µL
July 21st
Lisza
Made glycerol stocks of the 4 pTet-EYFP ligations
Kirsten, Alix
Mini-prep of pTet-EYFP (3 &4)x2 followed protocol according to Qiagen.
Analytic restriction digest of above mini-preps
Control used mini prepped pTet from June 24.
Added (in order) to 1.5mL micro centrifuge tubes:
- 12µL water
- 2 µL buffer tango
- 5 µL DNA
- 0.5µL XbaI
- Total: 20 µL
Megan, Kirsten, Alix, Lisza
Made an 80mL agarose gel for large rig
0.8g agarose
80mL 1x TAE
Lanes
- Ladder 1Kb
- Blank
- pTet unrestricted
- pTet undigested
- rxn III-1 undigested (pTet-EYFP)
- rxn III-1 digest
- rxn III-2 undigested
- rxn III-2 digest (XbaI)
- blank
- rxn IV-1 undigested
- rxn IV-1 digest (XbaI)
- rxn IV-2 undigested
- rxn IV-2 digest (XbaI
Ran at 120V for 40 min( in @ 5:45) stained in ethidium bromide for 10 20 min.
Picture:
Lisza
Made glycerol stocks of maxiprep cultures:
3 lumazine
2 Arg N-term
1 Arg c-term
Spun down cultures
Lumazine-1=1.35g
Lumzine-2= 1.31g
Lumazine-3=1.29g
Arg c-term=1.26g
Arg n-term-1=1.43g
Arg n-term-2=1.12g
July 23
Did a colony screen PCR:
24 colonies from July 14th DH5α Amp
AB+MC+MT plates labeled 10-32
With picked colonies transferred bacteria into a 1.5mL tube with 300µL milliQ water. With same toothpick inoculated 5mL (Amp 5mL) grown overnight at 37°C. Put 1.5L tubes onto heat block at 95°C for 7 min. Centrifuged at full speed for 1 min. Used 5µL supernatant for PCR survey.
Reactions: control with EYFP
- 10.4µL water (14.9 µL control)
- 2 µL Taq buffer
- 0.4 µL dNTPs
- 1 µL VR primer
- 1 µL VFZ primer
- 0.2 µL Taq polymerase
- 5 µL template (0.5 µL control)
Cycle: Colony58
Note: tube 11 contained both samples 11 and 12
July 24th
1% agarose gel at 100V of colony PCR
Lanes:
- 1 kb Ladder (8 µL)
- Control
- Colony 10 (10 µL sample+2 µL dye)
- Colony 11/12
- Colony 13
- Colony 14
- Colony 15
- Colony 16
- Colony 17
- Colony 18
- Colony 19
- Colony 20
- Colony 21
- Colony 22
- Colony 23
- Colony 24
- Colony 25
- Colony 26
- Colony 27
- Colony 28
- Colony 29
- Colony 30
- Colony 31
- Colony 32
- Colony 33
- 1 Kb ladder (8 µL)
PCR was successful, but had negative colonies. No pTet EYFP biobrick, only pTet was in the plasmid
July 27
Fan & Lisza
Preparative restriction digest of pTet-EYFP:
- 50µL reactions
- 40 µL DNA
- 5 µLbuffer
- 0.5 µL RE1
- 0.5 µL RE@
- 4 µL milliQ water
pTet:
EYFP:
Incubated at 37°C for 2 hours
Kirsten, Lisza, Mackenzie
Purified double digested pTet using QiaQuick PCR purification kit. First run through eluted DNA with 50 µL water then done again and eluted with 30 µL elution buffer. Into -20°C fridge
Gel Extraction of EYFP
Weight of tube: 1.0973g
Weight of tube+gel: 1.9759g
Weight gel: 0.8786
Transferred to falcon tube
Preformed gel extraction according to Qiaquick Gel Extraction kit. Eluted with 30 µL of elution buffer.
August
September
October
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