Team:Tec-Monterrey/Notebook
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July
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Week 3: 9-15
Pre-Interlab: Transformation Practice (p. 25, 07/13/18)
Antibiotic stock solutions (Carlos)
- Ampicillin 100 mg/ml
- Chloramphenicol 35 mg/ml
- Kanamycin 35 mg/ml
LB Medium (without agar) (Jesús)
- 25 g/L, 500 ml of medium
LB Medium (with agar) (Jesús)
- 25 g/L, 500 ml of medium
- 15 g/L of agar, 500 ml of medium
Medium LB and silica spheres are put inside autoclave
- 121°C for 15 minutes
- Materials left inside fume hood, with 15 min. of UV light
- Bain-marie prepared, 42°C
Pre-Interlab: Transformation Practice (p. 25, 07/13/18)
Transformations (Samantha)
- 4 Petris, 23K + antibiotic, 8P + antibiotic, 23K w/o antibiotic, competent w/o antibiotic
- Positive control: 8P, dish 3, kit 2018: BBa_I20270 (GFP)
- 1: 23K, dish 3, kit 2018: BBa_I763007 (RFP)
DNA from plates to Eppendorfs
- 1: 50 μL CC, 1 μL 23K
- 2: 50 μL CC, 1 μL 23K
- 3: 50 μL CC, 1 μL 8P
- 4: 50 μL CC
Plates (Victor)
- 20 ml LB with agar for all plates
- 20 μL of Chloramphenicol when needed
Heat Shock (Samantha)
- 1-4: 1 minute, 42°C, then 5 minutes on ice + 200 μL LB to each tube
Note: iGEM protocol says 450 ml of SOC, but instead we used 200 μL of LB
Incubations
- 37°C and 220 rpm, during 1 hour (6:03-7:03 pm)
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Week 4: 16-22
Interlab: Phase 1 and Preparations (p. 26-27, 07/16/18)
SOC medium preparation (NCb BioLabs, Thermo Fisher)
- 2% Tryptone
- 0.5% yeast extract
- 10 mM NaCl
- 2.5 mM KCl
- 10 mM MgCl2
- 10 mM MgSO4
- 20 mM Glucose
SOB medium preparation
- 2% Tryptone
- 0.5% yeast extract
- 10 mM NaCl
- 2.5 mM KCl
- 10 mM MgCl2
- 10 mM MgSO4
Buffer CCMB80 1L preparation
- 10 mM KOAc ph 7.0
- 80 mM CaCl2
- 20 mM MnCl2
- 10 mM MgCl2
Growth
- In 200 ml of SOC
Notes: iGEM protocol says 250 ml SOB, instead 200 ml were used
There’s less overnight, so initial absorbance will be less than 0.1
Optical density
- 3:00 pm, 0.3
- 3:52 pm, 0.465
Note: For buffer resuspension, 20 ml were used instead of 80 ml
RFP transformation after 16 hours
- 12 hours: 1-2 colonies, low expression
- 16 hours: 3-4 colonies, medium expression
Second competents batch
- Previous results were successful, so iGEM (buffer CCMB80) protocol will be repeated.
- Absorbance before the first resuspension: 0.482
- Resuspension in 20 ml of buffer
- Absorbance after resuspension: 0.444
Interlab: Plate 7 Transformations (p. 28-29, 07/17/18-07/18/18)
Devices
Wells
Eppendorf
Ctrl(-).BBa_R0040
7-2D, 7-4D, 7-21D, 6-20D, 2-6F
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Ctrl(+).BBa_I20270
7-2B, 7-4B, 7-21B, 6-20B, 3-8F
(+)
D1.BBa_J364000
7-2F, 7-4F, 7-21F, 6-20F
1
D2.BBa_J364001
7-2H, 7-4H. 7-21H, 6-20H
2
D3.BBa_J364002
7-2J, 7-4J, 7-21J, 6-20J
3
D4.BBa_J364003
7-2L, 7-4L
4
D5.BBa_J364004
7-2N, 7-4N
5
D6.BBa_J364005
7-2P, 7-4P
6
DNA Resuspension (Samantha)
- With respect to the wells on plate 7
Competent cells
- 50 μL to each Eppendorf duplicate
Resuspended DNA
- 2 μL were added to each Eppendorf duplicate
Colonies
1.1
1.2
1.3
2.1
2.2
2.3
3.1
3.2
3.3
4.1
4.2
4.3
D3
388
417
381
536
491
523
498
452
575
548
559
300
D4
71
167
213
223
200
197
255
242
165
116
269
281
D5
15
52
61
65
50
218
55
78
50
56
79
97
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August
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Week 2: 6-12
Overnights (p. 29, 08/07/18-08/08/18)
(Arnulfo)
BL21 Cas1Cas2 WT 1 colony
BL21 Cas1Cas2 WT 4 colonies
DH5⍺ Cas1Cas2 tags 4 colonies
DH5⍺ Cas1Cas2 tags 1 colony
DH5⍺ Cas1Cas2 WT colony
Note: First two overnights showed no growth, DNA plasmid
extraction protocol was conducted on the other 3 overnights.Minipreps (Carlos)
- Marked solutions protocol on kits was followed
- Low amounts of DNA were obtained from extraction
Minipreps Digestion (p. 31, 08/09/18)
(Arnulfo)
NEB Protocol: For plasmid DNA, Invitrogen protocol was followed.
- Step 4: Nomenclature mistake while tagging samples.
- Step 6: N9 was added and accidentally thrown out along with the filter
Competent Cell Test Kit (p. 32, 08/10/18)
(Carlos, Victor, Norma)
iGEM Transformation Interlab Test 2018. 2 μL of DNA on each tube to reach the same concentration proposed on the protocol.
- Competent BL21 Cells (10:20 pm), 20 minutes of overnights and buffer CCMB80
Note: OD was not measured at the beginning
Transformations (p. 32, 08/10/18)
- Resuspend DNA from kit with 10 μL of H2O (turns red)
- Place competent cells on ice (10 min)
- Pipette 50 μL of competent cells in a 1.5 ml tube + 1 μL of resuspended DNA
- Pipette 1 μL of control DNA in 2 ml tube (resuspending on ice)
- Close 1.5 ml tubes, mixing by inversion and incubating 30 min
- Heat shock, 42°C, 45 s
- Incubate on ice, 5 min
- Pipette 950 μL of SOC medium in each transformation
- Incubate at 37°C for 2 hours at 200-300 rpm
- Pipette 100 μL of each transformation on a Petri dish
- Centrifugate at 6800 g for 3 min. Throw out 800 μL of leftover
- Resuspend cells on 100 μL leftovers and pipette each transformation on Petris
- Incubate transformations overnight at 37°C
Overnights LB + Str (p. 33, 08/10/18)
(Carlos)
- 20 ml sterile LB + Str 15 μL (100 μg/μL stock)
- Cultivate on a handle, starting 3:00 pm
IDTE buffer solution
- Tris and EDTA calculations
LB Medium (with agar) (Sofía)
- LB: 25 g/L on 500 ml medium
- Agar: 15 g/L on 500 ml medium
Minipreps (p. 33-34, 08/11/18)
(Lizeth, Ana, Andrés)
Invitrogen Academic Lab Kit 2018
- Using Cas1Cas2 WT and Cas1Cas2 Tag
- DNA concentration using Nanodrop
- Digestion with Xba1, using NEB protocol
Bands between 4000 and 5000 bps were obtained, which match with plasmids.
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Week 3: 13-19
Plates with P1, P2 and Construct (p. 34, 08/13/18)
(Ana, María, Alan, Valeria)
Miniprep of construct
- Using Invitrogen Academic Lab Kit 2018
- Nanodrop was left for tomorrow
Transformations (p. 35, 08/14/18)
(Samantha, Victor)
- BL21 - Cas1Cas2Flag, 2 μL DNA
- BL21 - Cas1Cas2WT, 2 μL DNA
- 70 1 μL CCBL21 (Alan)
SOC medium preparation
- Openwetware protocol of SOB medium + sterilized glucose for SOC
Minipreps (p. 35-36, 08/16/18-08/17/18)
(Sofía, Arnulfo, Nora)
- Cobalt (P1), Test 1 and Test 2 (P1-1 & P1-2)
- Lead(P2), Test 1 and Test 2 (P2-1 & P2-2)
- Construct, Test 1 and Test 2 (C1 & C2)
- Total: 6 minipreps
Digestion (Ana, Jesús)
Electrophoresis (p. 37, 08/16/18-08/18/18)
(Alan)
- EtBr was handled according to safety protocols since first trial had some mistakes
Plasmid DNA Digestion
- Buffer 10x and BSA 100x
- Cas1 and Cas2 gel
Miniprep of RT-his Flag WT
- P1 in DH5⍺, Construct in DH5⍺, P1 in BL21
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Week 4: 20-26
BL21 Flag and RT-his Transformation (p. 37, 08/20/18)
- Bain-marie, 10 s
- Ice, 5 min, then incubation
- Plating of 100 μL, 800 μL removed from medium
Note: Incubation was done 35 min. After icing without SOC
Transformations (p. 38, 08/22/18)
(Adrián)
- BL21 - Flag(str) and RT-His (IDT)(AMP), WT
- BL21WT - RT-His (IDT)(AMP)
- NEB protocol will be used
Note: Bain-marie is now set at 42°C with “control temperature” button
Minipreps Digestion (p. 38, 08/24/18)
(Carlos)
NEB Protocol for the following plates:
- BL21 P1, DH5⍺ WT, DH5⍺ Flag, DH5⍺ P1, DH5⍺ Rt-His, DH5⍺ construct, BL21 WT
SOC medium preparation (400 ml)
Transformations (p. 39, 08/25/18)
(Roberto, Ana, Nora)
- BL21 - 50 μL
- Flag (streptomycin) - 5 μL
- RT (ampicillin) - 5 μL
Digestions (Jesús, Victor)
- NEB protocol, p. 38
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Week 5: 27-31
Overnights (p. 39, 08/27/18)
(Jesús, Victor)
- 8:00 pm, set on LB: BL21 WT + RT-His 1, BL21 WT + RT-His 2, BL21 His + Flag
- 8:30 am, few growth on overnights
- 9:25 am, inoculation on SOC, 15 ml
- 11:10 am, no significant growth on overnights nor on SOC
Streptomycin solution (p. 40, 08/29/18)
(Esteban, Sofía)
- 50 mg/ml, online protocols
Glycerol stocks
- 500 μL BL21 and glycerol 50%
Transformations
- RT-His (IDT)
BL21 Minipreps (p. 40, 08/29/18)
(Carlos, Nora)
- RT-His and BL21 - Cas1Cas2: Flag, both pellets were red
Nanodrop
- Band = 4711
Heatshok Transformation
- Different antibiotics will be used: 15 μL and 20 μL of strepto & ampicillin
- In order to analyze results and standardize antibiotic concentrations
Digestion and gels for minipreps
- NEB protocol, WT-RT
Minipreps (p. 41, 08/30/18-08/31/18)
(Ana, Arnulfo)
- DNA concentrations for BL21 RT-His, Bl212 Cas1Cas2 Flag, BL21 Cas1Cas2 WT and BL21 Cas1Cas2 WT RT-His
Digestion
- NEB protocol, WT-RT for 50 μL
Electrophoresis (Adrián)
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September
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Week 2: 3-9
Ligation and Transformation (p. 42, 09/04/18)
(Andrés, Samantha, Jesús, Sofía)
- NO3 CAM, 2 plates
- PO3 CAM, 2 plates
- Construct CAM, 2 plates
- BFP CAM, 1 plate
- Stop CAM, 1 plate
- RBS AMP, 1 plate
EDTA 0.5M and Tris-HCl 1M solutions were prepared
LB and SOC preparations (p. 42, 09/05/18)
(Andrés, Esteban, Sofía)
- LB + agar (300 ml)
- SOC medium (300 ml)
Ligation and Transformation
Competent cells and autoclaving (p. 42, 09/08/18)
(Alan, Samantha, Sofía)
- Small, medium and large tips were autoclaved
- Ligations from 09/05/18 were plated and incubated
Digestion Minipreps Data (Carlos)
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Week 3: 10-16
Minipreps (p. 38, 09/10/18)
DH5⍺ (NO3, PO4, Construct) and BL21 (NO3, PO4, Construct) with the kit
- 15 min on ice instead of 30 min
- 60 s on HS instead of 10 s
- 2 min on ice instead of 5 min
SOC medium preparation (400 ml)
Minipreps for Construct, NO3, PO4 (p. 44, 09/13/18)
- Construct: CD DH5⍺ 17.1, CB BL21 7.2
- NO3: ND1 14.6, ND2 16.5, NB1 7.3, NB2 12.9
- PO4: PD1 11.8, PD2 14.7, PB1 9.7, PB2 14.8
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Week 4: 17-23
TAE TX 50x (200 ml) (p. 44, 09/17/18)
- Tris-base, acetic acid and EDTA
- 50x was not prepared, stock was taken to do TAE 1x
- 2 min on ice instead of 5 min
SOC medium preparation (400 ml)
Digestion (p. 44, 09/18/18)
(Carlos)
- For CD, CB, PD1, PB1, PB2, ND1, ND2, NB1, and NB2
- For WT, RT-His and Flag
Overnights (p. 45-46, 09/19/18)
- Overnights with duplicates on plates: DH5⍺-PO3, DH5⍺-NO3, BL21-PO3, BL21-NO3, DH5⍺-Construct, DH5⍺-Construct
PCR Reaction
- Stock 100 μM
Minipreps and PCR Repetition (Nora, Victor)
- DNA dilution for each target and concentration
New Transformations Protocols (p. 46, 09/20/18)
- Cells on ice, 10 min
- 50 μL cells + 1-5 μL DNA
- Resuspend, put on ice 15 min, heat shock 60 s, 2 min on ice
- 950 μL SOC
- Incubate at 37°C, 60 min, 250 rpm
- Preheat plates and add antibiotic
Minipreps
- RT minipreps showed a very dense yellowish fluid, it should be centrifuged more time
Digestions
- RT, Flag 69.6, Flag 56.2
Cas1Cas2 Plates (p. 47, 09/22/18)
- For Flag, WT (DH5⍺)
- Summary of previous results
PCR Gel (Ana)
Minipreps
- DH5⍺ Cas1Cas2 Flag
- DH5⍺ Flag
- DH5⍺ WT
- BL21 RT-His
- BL21 RT-His
Overnight minipreps (p. 47-48, 09/23/18)
Minipreps (PO and NO3)
Cas and RT (Arnulfo, Adrián, Sofía)
PCR Dilutions
Digestions and Backbone
- NO3 50 μL
- PO4 25 μL
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Week 5: 24-30
Digestion (p. 48, 09/24/18)
(Nora, Ana)
- For RT-His
Enzyme Master Mix (50 μL)
Digest Plasmid Backbone
Ligation
Electrophoresis gel (p. 49-50, 09/25/18-09/26/18)
- A high amount of interference was observed in Nanodrop, λ < 230 nm, for minipreps from 23/09 and from 25/09
Gel extraction
- 1.2 mL for 400 mg on both procedures
Gel digestion
Electrophoresis gel (p. 50-51, 09/28/18-09/29/18)
- A high amount of interference was observed in Nanodrop, λ < 230 nm, for minipreps from 23/09 and from 25/09
Gel extraction
- 1.2 mL for 400 mg on both procedures
Gel digestion
SOC medium preparation (Esteban)
TAE 50x buffer preparation 250 ml (Esteban)
Digestion and overnights
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October
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Week 1: 1-7
Minipreps (p. 51-52, 10/01/18)
Transformations
- With construct and NO3
Digestion
Digestion (p. 53, 10/04/18)
- For RT-His, WT, and Flag
Minipreps (p. 54, 10/06/18)
(Esteban, Adrián)
- With NO3 (IPT)
- Stock 100 μL
Ligations IDT + 4B
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Week 2: 8-14
Inoculation Cas1Cas2 WT (p. 54, 10/10/18)
(Samantha)
- Agitation 37°C, 11:10 am
Transformations (Sofía, Samantha)
Digestion and ligation (Samantha, Andrés)
Western Blot (p. 55, 10/13/18)
Overnights and IPTG inductions
- BL21 Cas1Cas2 Tags
- BL21 Cas1Cas2 WT
- BL21 RT-His
- BL21 virgin
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Week 3: 15-17
WikiFreeze!
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