iGEM 2018 — Team Tec-Monterrey

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# Team:Tec-Monterrey/Notebook

![](https://static.igem.org/mediawiki/2018/e/e3/T--Tec-Monterrey--Page_Loader.gif)

* [Tec de Monterrey](https://2018.igem.org/Team%3ATec-Monterrey)
* Project
  + [Description](https://2018.igem.org/Team%3ATec-Monterrey/Description)
    [Design](https://2018.igem.org/Team%3ATec-Monterrey/Design)
    [Interlab](https://2018.igem.org/Team%3ATec-Monterrey/InterLab)
    [Notebook](https://2018.igem.org/Team%3ATec-Monterrey/Notebook)
    [Safety](https://2018.igem.org/Team%3ATec-Monterrey/Safety)
    [Demonstrate](https://2018.igem.org/Team%3ATec-Monterrey/Demonstrate)
    [Medals](https://2018.igem.org/Team%3ATec-Monterrey/Medals)
* Parts
  + [Parts](https://2018.igem.org/Team%3ATec-Monterrey/Parts)
    [Improved parts](https://2018.igem.org/Team%3ATec-Monterrey/Improve)
    [Parts list](https://2018.igem.org/Team%3ATec-Monterrey/PartsList)
* People
  + [Team](https://2018.igem.org/Team%3ATec-Monterrey/Team)
    [Collaborations](https://2018.igem.org/Team%3ATec-Monterrey/Collaborations)
    [Attribution](https://2018.igem.org/Team%3ATec-Monterrey/Attributions)
    [Sponsors](https://2018.igem.org/Team%3ATec-Monterrey/Sponsors)
* Human Practices
  + [Human Practices](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices)
    [Public & Educational Engagement](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement)
* Modeling
  + [Overview](https://2018.igem.org/Team%3ATec-Monterrey/Model)
    [D.E. System](https://2018.igem.org/Team%3ATec-Monterrey/Model)
    [Quasi-Steady State](https://2018.igem.org/Team%3ATec-Monterrey/Model)
    [Protein Production](https://2018.igem.org/Team%3ATec-Monterrey/Model)

![](https://static.igem.org/mediawiki/2018/7/76/T--Tec-Monterrey--ECoding_Menu_Logo_Still_200.png)

PROJECT
PARTS
HUMAN PRACTICES
MODELING
PEOPLE

* [Description](https://2018.igem.org/Team%3ATec-Monterrey/Description)

  [Overview](https://2018.igem.org/Team%3ATec-Monterrey/Description#overview)

  [System](https://2018.igem.org/Team%3ATec-Monterrey/Description#system)

  [References](https://2018.igem.org/Team%3ATec-Monterrey/Description#references)

* [Design](https://2018.igem.org/Team%3ATec-Monterrey/Design)

  [Background](https://2018.igem.org/Team%3ATec-Monterrey/Design#background)

  [Module 1](https://2018.igem.org/Team%3ATec-Monterrey/Design#module1)

  [Module 2](https://2018.igem.org/Team%3ATec-Monterrey/Design#module2)

  [System](https://2018.igem.org/Team%3ATec-Monterrey/Design#system)

  [Adaptation](https://2018.igem.org/Team%3ATec-Monterrey/Design#adaptation)

* [InterLab](https://2018.igem.org/Team%3ATec-Monterrey/InterLab)

  [Procedure](https://2018.igem.org/Team%3ATec-Monterrey/InterLab#procedure-interlab)

  [Results](https://2018.igem.org/Team%3ATec-Monterrey/InterLab#results-interlab)

  [Conclusion](https://2018.igem.org/Team%3ATec-Monterrey/InterLab#conclusion-interlab)

* [Notebook](https://2018.igem.org/Team%3ATec-Monterrey/Notebook)

  [Logbook](https://2018.igem.org/Team%3ATec-Monterrey/Notebook#logbook)

  [Protocols](https://2018.igem.org/Team%3ATec-Monterrey/Notebook#protocols)

  [Safety](https://2018.igem.org/Team%3ATec-Monterrey/Safety)

  [Biosafety](https://2018.igem.org/Team%3ATec-Monterrey/Safety#biosafety)

  [Our Lab](https://2018.igem.org/Team%3ATec-Monterrey/Safety#our-lab)

  [Project Safety](https://2018.igem.org/Team%3ATec-Monterrey/Safety#project-safety)

* [Demonstrate](https://2018.igem.org/Team%3ATec-Monterrey/Demonstrate)

  [Medals](https://2018.igem.org/Team%3ATec-Monterrey/Medals)

* [Parts](https://2018.igem.org/Team%3ATec-Monterrey/Parts)

  [Overview](https://2018.igem.org/Team%3ATec-Monterrey/Parts#overview)

  [Basic Parts](https://2018.igem.org/Team%3ATec-Monterrey/Parts#basic_parts)

  [Composite Parts](https://2018.igem.org/Team%3ATec-Monterrey/Parts#composite_parts)

  [Collection](https://2018.igem.org/Team%3ATec-Monterrey/Parts#parts_collection)

* [Improved Parts](https://2018.igem.org/Team%3ATec-Monterrey/Improve)

  Previous Parts

  Improved Parts

* [Parts List](https://2018.igem.org/Team%3ATec-Monterrey/PartsList)

* [Team](https://2018.igem.org/Team%3ATec-Monterrey/Team)

  [Header](https://2018.igem.org/Team%3ATec-Monterrey/Team#page-top)

  [Team Members](https://2018.igem.org/Team%3ATec-Monterrey/Team#teamMembers)

  [PI 1](https://2018.igem.org/Team%3ATec-Monterrey/Team#pi1)

  [PI 2](https://2018.igem.org/Team%3ATec-Monterrey/Team#pi2)

  [Instructor](https://2018.igem.org/Team%3ATec-Monterrey/Team#instructor)

  [Advisors](https://2018.igem.org/Team%3ATec-Monterrey/Team#advisors)

  [Collaborators](https://2018.igem.org/Team%3ATec-Monterrey/Team#collaborators)

* [Collaborations](https://2018.igem.org/Team%3ATec-Monterrey/Collaborations)

  [Collabs](https://2018.igem.org/Team%3ATec-Monterrey/Collaborations#collabs)

  [Meetups](https://2018.igem.org/Team%3ATec-Monterrey/Collaborations#meetups)

* [Attributions](https://2018.igem.org/Team%3ATec-Monterrey/Attributions)

  [What we have done](https://2018.igem.org/Team%3ATec-Monterrey/Attributions#tec-monterrey-work)

  [General Support](https://2018.igem.org/Team%3ATec-Monterrey/Attributions#general-support)

  [HP Support](https://2018.igem.org/Team%3ATec-Monterrey/Attributions#HP-support)

  [Design/Wiki](https://2018.igem.org/Team%3ATec-Monterrey/Attributions#design-wiki)

  [Fund raising](https://2018.igem.org/Team%3ATec-Monterrey/Attributions#fund-raising)

  [Thanks](https://2018.igem.org/Team%3ATec-Monterrey/Attributions#thanks)

* [Sponsors](https://2018.igem.org/Team%3ATec-Monterrey/Sponsors)

* [Human Practices](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices)

  [Header](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices#page-top)

  [Intro](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices#introduction-HP)

  [Action Plan](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices#action-HP)

  [Journey](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices#journey-HP)

  [Integrated](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices#integrated-HP)

  [World](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices#world-HP)

* [Public & Educational Engagement](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement)

  [Magazine articles](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#magazine)

  [Presentations](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#presentation)

  [Social Media](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#social)

  [Congress](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#congress)

  [Tour](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#tour)

  [Workshops](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#workshops)

  [Seminar](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement#seminar)

* [Overview](https://2018.igem.org/Team%3ATec-Monterrey/Model)

  [Introduction](#page-top)

  [Biological System](#page-top)

* [D.E. System](https://2018.igem.org/Team%3ATec-Monterrey/Model)

  [Kinetic Diagram](#page-top)

  [Equations](#page-top)

  [Parameters](#page-top)

* [Quasi-Steady State](https://2018.igem.org/Team%3ATec-Monterrey/Model)

  [Equations](#page-top)

* [Protein Production](https://2018.igem.org/Team%3ATec-Monterrey/Model)

  [Equations](#page-top)

  [Experimentation](#page-top)

  [Parameters](#page-top)

* [Header](#page-top)
* [Logbook](#logbook)
* [Protocols](#protocols)

Logbook

* July

  + 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)
  + 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 | (-) |
    | 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 |
* August

  + 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.

    ---
  + 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
  + 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
  + 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)
* September

  + 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)
  + 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, NO****3****, PO****4** (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
  + 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 NO****3****)**

    **Cas and RT** (Arnulfo, Adrián, Sofía)

    **PCR Dilutions**

    **Digestions and Backbone**

    - NO3 50 μL
    - PO4 25 μL
  + 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**
* October

  + 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**
  + 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
  + Week 3: 15-17

    # WikiFreeze!

Protocols

**Note:**  Even though we know that these protocols work, you should always compare them with other reliable sources before you conduct the experiment.

Reactant Preparation and Pipetting Techniques

Competent Cells

Bacterial Transformation

Restriction analysis

Miniprep

DNA Gel Electrophoresis

Gel Extraction and Ligation

SDS Page Electrophoresis Protocol

#### Contact Us

Av. Eugenio Garza Sada 2501

64849 Monterrey, Mexico

#### Sponsors

* ![CECROPIA](https://static.igem.org/mediawiki/2018/5/56/T--Tec-Monterrey--CECROPIA.png)
* ![CONANP](https://static.igem.org/mediawiki/2018/a/a2/T--Tec-Monterrey--CONANP.png)
* ![Concretos Modernos del Sur](https://static.igem.org/mediawiki/2018/4/43/T--Tec-Monterrey--Concretos.png)
* ![GDX Holdings](https://static.igem.org/mediawiki/2018/8/89/T--Tec-Monterrey--GDX.png)
* ![GenScript](https://static.igem.org/mediawiki/2018/e/e9/T--Tec-Monterrey--GenScript.png)
* ![IDT](https://static.igem.org/mediawiki/2018/3/37/T--Tec-Monterrey--IDT.png)
* ![Ladesa](https://static.igem.org/mediawiki/2018/8/8d/T--Tec-Monterrey--Ladesa.png)
* ![NEB](https://static.igem.org/mediawiki/2018/5/5e/T--Tec-Monterrey--NEB.png)
* ![SnapGene](https://static.igem.org/mediawiki/2018/3/3c/T--Duesseldorf--snapgene.png)
* ![Opnetrons](https://static.igem.org/mediawiki/2018/2/24/T--Tec-Monterrey--Opentrons.png)
* ![NEB](https://static.igem.org/mediawiki/2018/f/f2/T--Tec-Monterrey--Amexcid_Logo.png)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tec-Monterrey/Experiments

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3ATec-Monterrey)
[HOME](https://2018.igem.org/Team%3ATec-Monterrey)

TEAM

[Team Members](https://2018.igem.org/Team%3ATec-Monterrey/Team)
[Collaborations](https://2018.igem.org/Team%3ATec-Monterrey/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ATec-Monterrey/Description)
[Design](https://2018.igem.org/Team%3ATec-Monterrey/Design)
[Experiments](https://2018.igem.org/Team%3ATec-Monterrey/Experiments)
[Notebook](https://2018.igem.org/Team%3ATec-Monterrey/Notebook)
[InterLab](https://2018.igem.org/Team%3ATec-Monterrey/InterLab)
[Model](https://2018.igem.org/Team%3ATec-Monterrey/Model)
[Results](https://2018.igem.org/Team%3ATec-Monterrey/Results)
[Demonstrate](https://2018.igem.org/Team%3ATec-Monterrey/Demonstrate)
[Improve](https://2018.igem.org/Team%3ATec-Monterrey/Improve)
[Attributions](https://2018.igem.org/Team%3ATec-Monterrey/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ATec-Monterrey/Parts)
[Basic Parts](https://2018.igem.org/Team%3ATec-Monterrey/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ATec-Monterrey/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ATec-Monterrey/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ATec-Monterrey/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ATec-Monterrey/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ATec-Monterrey/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ATec-Monterrey/Hardware)
[Measurement](https://2018.igem.org/Team%3ATec-Monterrey/Measurement)
[Model](https://2018.igem.org/Team%3ATec-Monterrey/Model)
[Plant](https://2018.igem.org/Team%3ATec-Monterrey/Plant)
[Software](https://2018.igem.org/Team%3ATec-Monterrey/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Tec-Monterrey)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tec-Monterrey/Results

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3ATec-Monterrey)
[HOME](https://2018.igem.org/Team%3ATec-Monterrey)

TEAM

[Team Members](https://2018.igem.org/Team%3ATec-Monterrey/Team)
[Collaborations](https://2018.igem.org/Team%3ATec-Monterrey/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ATec-Monterrey/Description)
[Design](https://2018.igem.org/Team%3ATec-Monterrey/Design)
[Experiments](https://2018.igem.org/Team%3ATec-Monterrey/Experiments)
[Notebook](https://2018.igem.org/Team%3ATec-Monterrey/Notebook)
[InterLab](https://2018.igem.org/Team%3ATec-Monterrey/InterLab)
[Model](https://2018.igem.org/Team%3ATec-Monterrey/Model)
[Results](https://2018.igem.org/Team%3ATec-Monterrey/Results)
[Demonstrate](https://2018.igem.org/Team%3ATec-Monterrey/Demonstrate)
[Improve](https://2018.igem.org/Team%3ATec-Monterrey/Improve)
[Attributions](https://2018.igem.org/Team%3ATec-Monterrey/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ATec-Monterrey/Parts)
[Basic Parts](https://2018.igem.org/Team%3ATec-Monterrey/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ATec-Monterrey/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ATec-Monterrey/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ATec-Monterrey/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ATec-Monterrey/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ATec-Monterrey/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ATec-Monterrey/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ATec-Monterrey/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ATec-Monterrey/Hardware)
[Measurement](https://2018.igem.org/Team%3ATec-Monterrey/Measurement)
[Model](https://2018.igem.org/Team%3ATec-Monterrey/Model)
[Plant](https://2018.igem.org/Team%3ATec-Monterrey/Plant)
[Software](https://2018.igem.org/Team%3ATec-Monterrey/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Tec-Monterrey)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)