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)

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)

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

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.

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

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

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)

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)

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

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

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

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

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

WikiFreeze!