Difference between revisions of "Team:Tec-Monterrey/Notebook"

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