Team:Lethbridge/Notebook

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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.

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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.

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

1. Ladder 1Kb
2. Blank
3. pTet unrestricted
4. pTet undigested
5. rxn III-1 undigested (pTet-EYFP)
6. rxn III-1 digest
7. rxn III-2 undigested
8. rxn III-2 digest (XbaI)
9. blank
10. rxn IV-1 undigested
11. rxn IV-1 digest (XbaI)
12. rxn IV-2 undigested
13. 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. 1 kb Ladder (8 µL)
2. Control
3. Colony 10 (10 µL sample+2 µL dye)
4. Colony 11/12
5. Colony 13
6. Colony 14
7. Colony 15
8. Colony 16
9. Colony 17
10. Colony 18
11. Colony 19
12. Colony 20
13. Colony 21
14. Colony 22
15. Colony 23
16. Colony 24
17. Colony 25
18. Colony 26
19. Colony 27
20. Colony 28
21. Colony 29
22. Colony 30
23. Colony 31
24. Colony 32
25. Colony 33
26. 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:

• RE1=pstI
• RE2=speI

EYFP:

• RE1=pstI
• RE2=xbaI

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.

July 28

Kirsten, Alix, Lisza

Ligation of pTet and EYFP

Reaction 1

• 1.55µL of water
• 2.5 µL 10x buffer
• 5 µL vector (pTet)
• 14.7 µL insert (EYFP)
• 1.25 µLT4 DNA ligase
• 25 µL total

Reaction 2 (Control)

• 16.25µL of water
• 2.5 µL 10x buffer
• 5 µL vector (pTet)
• 0.0µL insert (EYFP)
• 1.25 µLT4 DNA ligase
• 25 µL total

Incubate at room temperature for 2 hours then transform

Restriction Digest of Lumazine 1&2, C-terminal Arg tag, N-terminal Arg tag 1&2.

• 14.6µL of water
• 2.5 µL 10x buffer tango
• 4 µL DNA
• 0.4µ EcoRI
• 21 µL total

Incubate at 37°C for 2 hours in water bath.

July 28

Fan

Did a transformation with ligation reactions plus a water control.

July 29

Running gel of the PCR for EYFP and CFP with c-terminal fusion tags and the restriction digests of n-terminal, c-terminal ARG tags and the lumazine gene.

Loading 6µL ladder, 5µL sample+1µL dye

Lane:

1. 1kb DNA ladder
2. CFP amplicon
3. EYFP amplicon
4. Empty
5. N-terminal #1 (EcoRI digestion)
6. N-terminal #2 (EcoRI digestion)
7. C-terminal (EcoRI digestion)
8. Lumazine # 1 (EcoRI digestion)
9. Lumazine # 2 (EcoRI digestion)

Did colony PCR of the 5 colonies from the EYFP-1 plater, 5 of the EYFP-2 plate, 4 of the pTet-1, 4 of the pTet-2, one negative control and one positive (EYFP plasmid). Followed protocol of July 23.

Megan, Lisza, McKenzie

Purify maxipreps using kit. Finished 60 µL of PCR samples with a 5 buffer to 1 PCR sample ratio. Buffer amount = 300 µL. Followed protocol in kit.

Ran a gel with ladder, 3 wells for PCR, 3 double wells for extraction

July 30

Lisza

PCR for fusion c-terminal suffix

Control: no template DNA

• 1.4 µL milliQ water
• 4 µL buffer (5x Phusion)
• 0.4 µL 10mM dNTPs
• 1 µL sense
• 1 µL antisense
• 0 µL DNA
• 0.2 µL phusion polymerase

One for CFP and one for EYFP:

• 11.4µL milliQ water
• 4 µL buffer (5x Phusion)
• 0.4 µL 10mM dNTPs
• 1 µL sense (prefix primer)
• 1 µL antisense (antisense FP Fusion c-term tag)
• 2 µL DNA
• 0.2 µL Phusion polymerase

One for CFP and one for GFP:

• 28.5µL milliQ water
• 10 µL buffer (5x Phusion)
• 1 µL 10mM dNTPs
• 2.5 µL sense (prefix primer)
• 2.5 µL antisense (antisense FP Fusion c-term tag)
• 2 µL DNA
• 0.5 µL Phusion polymerase

Control: no polymerase (water= 29µL)

Roxanne

1% agarose gel in TAE of the colony PCR

E1= plate 1 of EYFP ligation

E2= plate 2 of EYFP ligation

(-)= no template

(+)= EYFP/no pTet

P1=plate 1 of pTet control ligation

P2=plate 2 of pTet control ligation

Lane

1. Ladder
2. E1-1
3. E1-2
4. E1-3
5. E1-4
6. E1-5
7. E2-1
8. E2-2
9. E2-3
10. E2-4
11. E2-5
12. +
13. –
14. P1-1
15. P1-2
16. P1-3
17. P1-4
18. P2-1
19. P2-2
20. P2-3

Completed the maxipreps of pBAD, TetR, pLacI, mRBS,sRBS and Lumazine 2. They were left at isopropanol precipitation.

Restriction Digest

dT-EcoRI & XbaI

pTet-SAP

pSB1A3-1 -EcoRI, Spe1

n-terminal tag-XbaI, PstI

Lumazine- XbaI, Spe1

pSB1A3-2- XbaI, PstI

C-terminal tag- EcoRI, Spe1

Prefix-N-terminal tag//gene Gene//c-terminal-suffix

July 30

Ran a 1x agarose gel of PCR products from earlier.

Lane:

1. 1kb ladder
2. Control
3. CFP-phusion
4. EYFP-phusion
5. CFP-econo taq
6. EYFP-econo taq

The PCR worked

July 31

Mackenzie + Lisza

Gel extraction of c-term tag, n-term tag and lumazine.

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August

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September

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October

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