NoteBook
Day 1, Monday 04/06
People In Lab: Amalia, Carla, Daniel, Jasmeen, Nina
Agenda:
LB media
LB + agar plates
Aliquoted autoclaved SOC
70% EtOH
Equipment used:
Autoclave
Bunsen burner
Protocol: iGEM protocols used for LB, LB+agar, SOC (altered to accommodate MgCl2·6H2O because we didn't have anhydrous version, thus, for 1L solution, 2.17g was needed).
Next Steps:
Get MG1655ΔlacI cells from Christian and streak onto LB plate
Transform arg1 and pSB1C3+RFP (iGEM competence test plasmid) into DH10β
Make MG1655ΔlacI glycerol stocks
Make LB+CAM and LB+KAN plates
Day 2, Tuesday 05/06
People In Lab: Carla
Agenda:
KAN plates
CAM plates
Equipment used:
Autoclave
Bunsen burner
Protocol: iGEM protocol used for LB+agar.
Calculation for antibiotics to be added to LB media for plating
Chloramphenicol
C1V1= C2V2
C1= stock concentration = 25mg/mL
C2 = working concentration = 25μg/mL
(25000μg/mL)(V1) = (25μg/mL)(500mL)
V1= 0.5mL
Kanamycin
C1V1= C2V2
C1= stock concentration = 10mg/mL
C2 = working concentration = 50μg/mL
(10000μg/mL)(V1)
= (50μg/mL)(500mL)
V1=
2.5mL
Next Steps:
Obtain overnight culture of MG1655ΔlacI in the morning left for us in the shaker in room 318
Day 3, Wednesday 06/06
People In Lab:Amalia, Carla, Daniel, Jasmeen, Monica, Nina, Tashi
Agenda:
Transform pET28a+arg1 and pSB1C3+RFP (iGEM competence test plasmid) into DH10β
Streak MG1655ΔlacI onto LB plate
Equipment used:
ThermoMixer
Bunsen Burner
37°C incubator in room 301
Protocol: iGEM protocols used for transformation.
Transformation
Transformation 1: pSB1C3+RFP(10pg/μL) in DH10β: 100μL plated on LB+CAM
Transformation 2: pSB1C3+RFP (10pg/μL)in DH10β: 100μL plated on LB+CAM
Transformation 3: pET28a+arg1 in DH10β: 100μL plated on LB+KAN
Transformation 4: pET28a+arg1 in DH10β: 100μL plated on LB+CAM
Negative control: 100μL DH10β plated on LB+CAM and LB+KAN
For each transformation, 50μL of cells and 2μL of plasmid, and 250μL of SOC were used
Next Steps:
Start interlab
Day 4, Thursday 07/06
People In Lab: Ahmed, Amalia, Daniel, Jasmeen, Nina
Agenda:
Observed transformed cells
pSB1C3+RFP (10pg/μL) in DH10β
pET28a+arg1 in DH10β
Observe streaked plate (LB+MG1655ΔlacI)
Make overnight cultures from transformed cells (37°C)
3 LB+CAM+DH10β+pSB1C3+RFP
3 LB+KAN+DH10β+pET28a+arg1
1 LB (negative control)
Transform interlab plasmids into DH5α
10 nuclease-free water aliquots
Equipment used:
Thermomixer
Bunsen Burner
37°C incubator in room 301
Protocol:Interlab protocols used for interlab transformation.
Interlab transformation
resuspend plasmids from plate 7 to a concentration of 200-300pg/μL
1μl plasmid used: 2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P
50μL of competent cells used (DH5α)
50μL of cells plated on LB+CAM
2J was not incubated, has to be transformed again
Observations:
Picture of plates are shown below.
100μl
transformation pET28a+arg1 in DH10β on LB+KAN
100μl transformation pSB1C3+RFP in DH10β on LB+KAN
100μl negative control (DH10β no plasmid) on LB+KAN and LB+CAM
Streaked MG1655ΔlacI on LB
Next Steps:
Miniprep pSB1C3+RFP and pET28a+arg1 from overnight cultures
Interlab calibration #1: OD600 Reference point - LUDOX Protocol
Day 5, Friday 08/06
People In Lab:Ahmed, Amalia, Daniel, Jasmeen, Nina, Tashi
Agenda:
Observe interlab transformations
Miniprep pSB1C3+RFP and pET28a+arg1 from overnight cultures
Nanodrop
Send miniprepped pSB1C3+RFP and pET28a+arg1 to Ranomics to add UNS2 and UNS3 to pSB1C3+RFP and fix illegal cut sites in pET28a+arg1
Interlab calibration #1: OD600 Reference point - LUDOX Protocol
Equipment used:
Thermomixer
Bunsen burner
37°C Incubator in room 301
Centrifuge
Nanodrop
Plate reader
Protocol: Protocol from miniprep kit used for miniprep. Interlab protocol used for Interlab calibration #1: OD600 Reference point - LUDOX Protocol.
Miniprep of pSB1C3+RFP and pET28a+arg1
10mL of each overnight culture used
60μL of nuclease-free water used for each elution
Sending miniprepped pSB1C3+RFP and pET28a+arg1 to Ranomics
(Concentration after nanodrop)(Volume sent) = (mass sent)
pSB1C3+RFP: (39.3ng/μL)(51μL) = 2004.3ng
pET28a+arg1: (142.0ng/μL)(21μL) = 2982ng
Observations:
Interlab Transformation of 2F, 2H, 2N on LB+CAM
Interlab Transformation of 2B, 2D, 2L, 2P on LB+CAM
Interlab calibration #1: OD600 Reference point - LUDOX Protocol absorbance values
*Refer to interlab page on wiki*
Next Steps:
Transform 2J plasmid into DH5α
Make LB+KAN+CAM plates
Make PBS (dilute from stock in fridge or make from scratch)
Transform pET28a+arg1and pSB1C3+RFP into MG1655ΔlacI
Interlab calibration #2: Particle Standard Curve - Microsphere Protocol
Day 1, Monday 11/06
People In Lab:Amalia, Carla, Daniel, Jasmeen, Nina, Tashi
Agenda:
Transform 2J interlab plasmid into DH5α and plate on LB+CAM
LB+CAM plates
Interlab calibration #2: Particle Standard Curve - Microsphere Protocol
Electroporate MG1655ΔlacI
Transform pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI
Make and autoclave PBS for interlab calibration #3
Autoclave double distilled water
Equipment used:
Autoclave
Bunsen burner
Plate reader
Thermomixer
37°C incubator in room 301
Electroporator
Protocol: iGEM protocols used for transformation, electroporation, LB+agar. Interlab protocol used for Interlab calibration #2: Particle Standard Curve - Microsphere Protocol.
Transformation of 2J plasmid
1μLof 2J plasmid used, 50μLof cells plated
Interlab calibration #2
200μLbead stock in wells A1, B1, C1, D1
100μLdouble distilled water in wells A2-A12, B2-B12, C2-C12, D2-D12
performed serial dilution by transferring 100μLeach time, until well A11, B11, C11, D11
used the plate reader to shake the plate at an amplitude of 3.5 for 7 seconds, and measure the absorbance at wavelength 600nm
Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI
2μLof pSB1C3+RFP(10pg/μL)
1μLofpET28a+arg1
plated 100μLon LB+CAM+KAN
plated 200μLon LB+CAM+KAN
plated 300μLon LB+CAM+KAN
plated negative control (100μLof untransformed MG1655ΔlacI)
Next Steps:
Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol
Interlab cell measurement protocol
Day 2, Tuesday 12/06
People In Lab: Amalia, Carla, Daniel, Nina
Agenda:
Second attempt at pSB1C3+RFP and pET28a+arg1 double transformation in MG1655ΔlacI, testing for incompatibility
Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol
Make overnight cultures from interlab transformation plates
Equipment used:
Bunsen burner
Plate reader
Electroporator
Shaker in room 315
37°C incubator in room 301
Protocol: iGEM protocols used for transformation by electroporation. Interlab protocol used for for Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol
Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI (electroporation)
Hypothesis: If we get growth on the positive control, LB+KAN, and LB+CAM plates with transformed cells, but not the double antibiotic plate, we can say that either the plasmids are incompatible, or the double antibiotic plate concentration is too high.
1.5μLof RFP (50pg/μL)
1.5μLof pET28a+arg1
plated 150μLof transformed cells on LB+CAM+KAN
plated 100μLof transformed cells on LB+CAM
plated 100μLof transformed cells on LB+KAN
plated 10μLof untransformed cells on LB+KAN (negative control)
plated 10μLof untransformed cells on LB (positive control)
Interlab calibration #3
serial dilution was performed and the plate was read
shaken 5s at amplitude = 2
excitation = 485nm
emission = 525nm
optimal gain used = 75
read from bottom of plate
The graph obtained from this experiment indicated an error, will repeat tomorrow
Interlab transformation overnight cultures
16 tubes with 5mL of LB and 5μLCAM
2 colonies were taken from each of the 8 plates (2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P)
tubes placed in the shaker overnight at 37° at 250rpm
Observations:
Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI
no colonies on negative control plate
no colonies on 100μL, 200μL, or 300μLplates
100μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI (negative control) on LB+CAM+KAN
100μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN
200μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN
300μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN
Potential reasons for results:
antibiotic concentration too high (50μL/mL KAN + 25μL/mL CAM)
MG1655ΔlacIcells may no longer be competent
left MG1655ΔlacIcells thawing in the ice for too long
electroporation might have failed
plasmids may not have been compatible
we could try sequential transformation (still have to consider antibiotic concentration of LB+KAN+CAM plate)
Next Steps:
Finish Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol
Cell growth, sampling, assay (interlab cell measurement)
Day 3, Wednesday 13/06
People In Lab:Amalia, Carla, Daniel, Nina
Agenda:
Redo Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol
Autoclave glass bottles, beads, tips, eppendorf tubes
Reculture the overnight cultures from the interlab transformation
Equipment used:
Autoclave
Bunsen burner
Plate reader
37°C shaker in room 315
Protocol: Used interlab protocol for interlab calibration.
Second attempt at Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol
serial dilution was performed and the plate was read
excitation = 485nm
emission = 525nm
optimal gain used = 90
read from bottom of plate
Reculturing the overnight cultures from the interlab transformation
16 tubes with 5mL of LB and 5μLCAM
100μLfrom each previous overnight culture placed in respective new tube (DH5α)
tubes slightly unscrewed and tape placed on lid for aeration in shaker
tubes placed in the shaker overnight at 37°C at 250rpm
Observations:
Second double transformation pSB1C3+RFP and pET28a+arg1in MG1655ΔlacI
LB+CAM+pSB1C3+RFP and pET28a+arg1
sparsely populated pink colonies
presence of some colonies
LB+KAN+pSB1C3+RFP and pET28a+arg1
no visible colonies
LB+KAN+CAM+pSB1C3+RFP and pET28a+arg1
no visible colonies
LB+KAN+nontrasformed cells (negative control)
no visible colonies
LB+nontransformed cells (positive control)
growth on entire plate
Second double transformation pSB1C3+RFP and pET28a+arg1 in MG1655ΔlacI
Potential reasons for these results:
colonies on LB+CAM+pSB1C3+RFP+pET28a+arg1) because the cells are still competent and were transformed with pSB1C3+RPF
no colonies on LB+KAN+pSB1C3+RFP and pET28a+arg1not because the cells are not competent, but rather the transformation (pET28a+arg1into MG1655ΔlacI) failed
no colonies on LB+KAN+CAM+pSB1C3+RFP+pET28a+arg1 because the backbones are not compatible
no growth on LB+KAN+non transformed cells (negative control) because there was no antibiotic resistance gene in the cells
we had growth on LB+non transformed cells (positive control) because the cells were viable and there was no selection in the plate against them
pET28a = KAN resistant - incompatible with pSB1C3 by group check
pSB1C3 = CAM resistant - higher copy number than pET28a
Next Steps:
Finish interlab cell measurement protocol
Day 4, Thursday 14/06
People In Lab:Ahmed, Amalia, Daniel, Monica, Nina, Tashi
Agenda:
Interlab cell measurement protocol
Autoclave P1000 tips
LB media
LB+CAM plates
Equipment used:
Autoclave
Plate reader
Shaker in autoclave room
Bunsen burner
37°C incubator in room 301
Protocol: iGEM protocols used for LB, LB+agar. Interlab protocols used for interlab OD and CFU measurements.
Observations:
Next Steps:
Count colonies on CFU cell measurement plates for interlab cell measurement protocol
Transform pET28a+arg1intoMG1655ΔlacI cells
Day 5, Friday 15/06
People In Lab:Ahmed, Amalia, Nina, Tashi
Agenda:
Check overnight plated cultures for interlab CFU count
Transformation of pET28a+arg1 into electrically competent MG1655ΔlacIcells
Observations:
Interlab CFU count
Miniscule colonies on some plates after ~11 hours of incubation at 37°C. Some plates did not have any colonies. Plates were put back in the incubator for a few more hours.
Equipment used:
37°C incubator in room 301
Shaker in room 315
Electroporator
Protocol: iGEM protocols used for transformation by electroporation.
Transformation of ARG1 in electrically competent MG1655ΔlacIcells
pET28a+arg1diluted from 39.3ng/mL to 1ng/mL
38.3μLnuclease free water + 1μLpET28a+arg1
transformed cells in cuvette placed in shaker at 37°C, 250rpm
3 plates
40μLof untransformed MG1655ΔlacIcells (negative control)
100μLof MG1655ΔlacItransformed with pET28a+arg1
200μLof MG1655ΔlacItransformed with pET28a+arg1
Next Steps:
Redo Calibration #2: Particle Standard Curve - Microsphere Protocol
Overnight culture of 2B (2 colonies) and 2D (2 colonies) in DH5α
If pET28a+arg1 transformation works
2 MG1655ΔlacI overnight cultures (LB+KAN+IPTG)
2 MG1655ΔlacI overnight cultures (LB+KAN)
streak a single MG1655ΔlacIcolony on an LB+KAN plate
Day 1, Monday 18/06
People In Lab: Amalia, Daniel, Jasmeen, Nina, Tashi
Agenda:
- LB+CAM plates
- Overnight culture of 2B (2 colonies) and 2D (2 colonies) (DH5α) for interlab
- 2 overnight cultures of LB+KAN+MG1655ΔlacI+IPTG
- 2 overnight cultures of LB+KAN+MG1655ΔlacI
- Streak plate pET28a+arg1 transformation on LB+KAN to get single colonies (MG1655ΔlacI)
- Autoclave microcentrifuge tubes
Equipment used:
- Autoclave
- Bunsen burner
- Shaker in room 315
Protocol: iGEM protocols used for LB+agar
Overnight cultures of colonies 2B+2D for interlab CFU
-4 LB+CAM tubes each with 5mL LB+5μL CAM
-2 colonies taken from 2B plate and inoculated
-2 colonies taken from 2D plate and inoculated
-all tubes placed in the shaker at 37°C at 250rpm
Serial dilution of IPTG
-dilution 1: 30μL of 1M IPTG stock in 10mL of LB = conc. 3mM
-10μL of dilution 1 in 10mL of LB = conc. 3μM
Overnight cultures of pET28a+arg1
-4 tubes
-2 with 5mL LB+IPTG+25μL KAN
-2 with 5mL LB+25μL KAN
-all tubes placed in the shaker at 37°C at 250rpm
Observations:
pET28a+arg1 transformation into MG1655ΔlacI
-No colonies on negative control plate or 100μL plate of transformed cells
-2 blobs on the 200μL plate of transformed cells
pET28a+arg1 transformation into MG1655ΔlacI negative control - 40μL untransformed cells
pET28a+arg1 transformation into MG1655ΔlacI 100μL transformed cells
pET28a+arg1 transformation into MG1655ΔlacI 200μL transformed cells
CFU colony count
Colony 1 |
Colony 2 |
|||||||
2B |
dilution 3 |
210 |
201 |
224 |
134 |
0 |
300 |
|
dilution 4 |
* |
* |
* |
* |
* |
* |
||
dilution 5 |
* |
* |
* |
* |
* |
* |
||
2D |
dilution 3 |
144 |
174 |
108 |
352 |
273 |
372 |
|
dilution 4 |
31 |
16 |
33 |
20 |
1 |
11 |
||
dilution 5 |
3 |
1 |
3 |
1 |
4 |
3 |
*plates were cracked because they were frozen
Next Steps:
- Redo interlab CFU protocol
- Let overnight cultures settle on bench (LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG) and (LB+KAN+MG1655ΔlacI+pET28a+arg1)
- Centrifuge pET28a+arg1 overnight cultures at 350g for 4 hours (LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG) and (LB+KAN+MG1655ΔlacI+pET28a+arg1)
- Streak out a single colony from the MG1655ΔlacI+pET28a+arg1 plate on an LB+KAN plate
Day 2, Tuesday 19/06
People In Lab: Amalia, Daniel, Nina, Tashi
Agenda:
- Continue interlab CFU protocol (up until plating)
- Centrifuge 1 overnight culture (LB+KAN+MG1655ΔlacI+IPTG) and 1 overnight culture (LB+KAN+MG1655ΔlacI) for 4 hours at 350g
- Keep 1 overnight culture (LB+KAN+MG1655ΔlacI+IPTG) and 1 overnight culture (LB+KAN+MG1655ΔlacI) on bench and observe
- Re-streak MG1655ΔlacI+pET28a+arg1 transformation on LB+KAN to get single colonies (was not incubated yesterday)
- Make new overnight culture of MG1655ΔlacI+pET28a+arg1
Equipment used:
- Bunsen burner
- Plate reader
- Shaker in autoclave room
- Centrifuge in room 301
- 37°C incubator in room 301
Protocol: Interlab protocol used for CFU experiement
Overnight culture of LB+KAN+MG1655ΔlacI+pET28a+arg1
-5mL LB+25μL KAN+incoulation from a blob from the LB+KAN+MG1655ΔlacI+pET28a+arg1 transformation plate
-tube placed in shaker at 37°C at 220rpm
Observations:
pET28a+arg1 induction
LB+KAN+MG1655ΔlacI+pET28a+arg1, LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG in centrifuge - cells pelleted at the bottom
-potential reason - too many gs
LB+KAN+MG1655ΔlacI+pET28a+arg1, LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG on bench - no separation
-potential reasons -arg1 not induced by IPTG?
-too hard flotation to see because there is no reporter
Centrifuged LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG (left )and LB+KAN+MG1655ΔlacI+pET28a+arg1 (right)
Next Steps:
- LB media
- LB+KAN plates
- Dilute overnight culture to OD=0.3
- Make IPTG dilution
- Induce LB+KAN+MG1655ΔlacI+pET28a+arg1 culture with 3μM IPTG for 22 hours (2 tubes)
- Make new LB+KAN+MG1655ΔlacI+pET28a+arg1 overnight culture for 22 hours (2 tubes)
- Count colonies for interlab CFU
- Streak out a single colony from the (MG1655ΔlacI) plate on an LB+KAN plate
- Make protocol for growth curve for dry lab
Day 3, Wednesday 20/06
People In Lab: Amalia, Daniel, Jasmeen, Nina, Tashi
Agenda:
- Make LB liquid media
- Make LB+KAN plates
- Count colonies for interlab CFU
- Streak out a single colony from the MG1655ΔlacI+pET28a+arg1 plate on an LB+KAN plate
- Induce LB+KAN+MG1655ΔlacI+pET28a+arg1 culture with 3μM IPTG for 22 hours (2 tubes)
- Make new LB+KAN+MG1655ΔlacI+pET28a+arg1 overnight culture for 22 hours (2 tubes)
Equipment used:
- Autoclave
- Bunsen burner
- Shaker in autoclave room
- 37°C incubator in room 301
- Spectrophotomter
Protocol: iGEM protocols used for LB+agar, interlab protocol used for CFU experiement
IPTG dilution #1 in LB
C1V1 = C2V2
C1 = 1M
C2 = 3mM
V2 = 10mL
V1 = 30μL
= take 30ul of stock and add to 10mL LB+KAN
IPTG dilution #2 in LB
C1V1 = C2V2
C1 = 3mM
C2 = 3uM
V2 = 25mL
V1 = 25μL
= take 25ul of dilution #1 and add to LB+KAN
pET28a+arg1 overnight culture
1:8 dilution, measure OD, OD = 0.25
0.25x8 = 2
C1V1 = C2V2
C1 = 2
C2 = 0.3
V2 = 25mL
V1 = 3.75mL
Total volume = 25mL
25mL - 3.75mL = 21.25mL
-21.25mL LB + KAN + 3.75mL overnight culture
-21.25mL LB + KAN + IPTG + 3.75mL overnight culture
Abs600 blank (LB+KAN+IPTG) = 0.00 (tared)
Abs600 (LB+KAN+IPTG+ARG1) = 0.127
Both tubes placed in shaker at 37°C at 220 rpm (one with IPTG and one without)
Observations:
CFU colony count #2
Colony 1 |
Colony 2 |
|||||||
2B |
dilution 3 |
180 |
138 |
40 |
77 |
78 |
91 |
|
dilution 4 |
6 |
16 |
8 |
10 |
8 |
4 |
||
dilution 5 |
2 |
3 |
2 |
1 |
0 |
1 |
||
2D |
dilution 3 |
215 |
150 |
149 |
83 |
115 |
274 |
|
dilution 4 |
45 |
28 |
28 |
30 |
14 |
28 |
||
dilution 5 |
2 |
2 |
2 |
0 |
4 |
4 |
Colony forming unit calculation (using Dilution 3 data)
Calculated for Interlab Form IV
(# of colonies) x (Final Dilution Factor) = CFU/mL
Final Dilution Factor = 8 x 104
Colony 1 |
Colony 2 |
- |
Colony 1 |
Colony 2 |
||
2B |
8.64 x 10^6 |
6.16 x 10^6 |
- |
2D |
1.72 x 10^7 |
6.64 x 10^6 |
1.104 x 10^7 |
6.24 x 10^6 |
- |
1.2 x 10^7 |
9.20 x 10^6 |
||
3.20 x10^6 |
7.28 x 10^6 |
- |
1.192 x 10^7 |
2.192 x 10^7 |
MG1655ΔlacI+pET28a+arg1 streak to get single colonies
Next Steps:
- LB+KAN plates
- Make KAN dilutions
- Order primers
- Order blue protein
Day 4, Thursday 21/06
People In Lab: Amalia, Daniel, Jasmeen, Nina
Agenda:
- Make LB + KAN plates
- Make KAN dilutions
- Make overnight culture of LB+KAN+MG1655ΔlacI+pET28a+arg1 for dry lab growth curve
- Order primers for pET28a+arg1
- Order blue protein
Equipment used:
- Bunsen burner
- Shaker in room 315
Protocol:
KAN dilutions
(800μL of nuclease-free water+200μL 50mg/mL KAN = 1mL 10mg/mL KAN) x3
Overnight culture of LB+KAN+MG1655ΔlacI+pET28a+arg1 for growth curve
-(15mL LB + 75μL KAN + incoulation from colony on newest MG1655ΔlacI+pET28a+arg1 plate) x3
-tube placed in shaker at 37°C at 220rpm
Observations:
MG1655ΔlacI+pET28a+arg1 streak to get clonal colonies
Next Steps:
- LB+KAN plates
- Make KAN dilutions
- Do digest and run gel
- Dry lab growth curve
- Order amilCP
Day 5, Friday 22/06
People In Lab: Amalia, Nina, Daniel, Tashi
Agenda:
- Make LB + KAN plates
- Make KAN dilutions
- LB+KAN+MG1655ΔlacI+pET28a+arg1 growth curve
- Order amilCP
Equipment used:
- Bunsen burner
- Shaker in autoclave room
- Plate reader
- Autoclave
Protocol: iGEM protocols for LB+agar and KAN dilutions
KAN dilutions
-80mL of 10mg/mL were made
-50 microcentrifuge tubes were filled with 1mL each and 30mL remains in a falcon tube in the fridge
LB+KAN+MG1655ΔlacI+pET28a+arg1 growth curve
-1:8 dilution made from each overnight culture (175mL LB+KAN and 25mL culture in well)
-200μl LB+KAN pipetted in well (blank)
-measure OD600
-subtract blank value, multiply by 8 to get original concentration (OD)
-dilute each overnight culture to get an OD of 0.1
-take OD again (0h timepoint)
-place the three replicates back in the shaker and measure again every hour (200μL)
Observations:
Volumes for LB+KAN+MG1655ΔlacI+pET28a+arg1 in growth curve cultures based on OD values of 1:8 dilutions-blank value multiplied by 8
Overnight culture 1 1:8 dilution OD without blank = 0.2209
Overnight culture 1 original OD = 1.3576
Replicate 1 LB+KAN = 23.158mL
Replicate 1 overnight culture = 1.841mL
Overnight culture 2 1:8 dilution OD without blank = 0.2167
Overnight culture 2 original OD = 1.324
Replicate 2 LB+KAN = 23.111mL
Replicate 2 overnight culture = 1.888mL
Overnight culture 3 1:8 dilution OD without blank = 0.2235
Overnight culture 3 original OD = 1.3784
Replicate 3 LB+KAN = 23.186mL
Replicate 3 overnight culture = 1.814mL
LB+KAN+MG1655ΔlacI+pET28a+arg1 in growth curve data
0 |
0.094 |
0.0942 |
0.1022 |
*0.0512 |
1 |
0.1808 |
0.1757 |
0.1713 |
|
2 |
0.255 |
0.2538 |
*0.4448 |
|
3 |
0.3391 |
0.3247 |
0.3332 |
|
4 |
0.3937 |
0.4075 |
0.4032 |
|
5 |
0.4539 |
0.4688 |
0.4326 |
|
6 |
0.4652 |
0.4797 |
0.4705 |
*Replicate 3 OD at 2h has an incorrect measurement. All 2h replicates were retested an hour later and they all had very similar values.
*All measurements shown already have the blank value subtracted from the raw value
Next Steps:
- PCR pET28a+arg1 with new primers
- Give Scott LB plate to streak with BL21
- LB+AMP plates (for gibson positive control)
- Order gibson
- Order amilCP