Team:ETH Zurich/lab/protocols

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<html><article id='materials'></html>
==Materials==
==Materials==
-
===LB medium===
+
 
 +
===NanoDrop 2000, UV-Vis Spectrophotometer===
 +
[[File:ETH Zurich 2014_NanoDrop.jpg|float|300px|thumb|'''Figure 1''' Our NanoDrop spectrophotometer]]
 +
 
 +
A Thermo Scientific NanoDrop® 2000 spectrophotometer was used to determine the concentrations of our plasmids after miniprep.
 +
 
 +
This microvolume spectrophotometer can be used to measure the concentration and purity of DNA, RNA or protein samples. Having a wide spectral range (190-840nm) allows for measuring a variety of samples types such as peptides, DNA, RNA, purified protein and even gold nanoparticles.
 +
Even for highly concentrated samples no dilutions are required. Results are obtained in less than 15 seconds from sample pipetting to wiping the pedestal clean. For the measurement only 0.5 – 2uL of the samples are needed.
 +
The software with an intuitive user interface allows for classical NanoDrop applications such as nucleic acids, protein A280, colorimetric assays and new user defined custom methods.
 +
 
 +
Please visit the [http://www.nanodrop.com/Productnd2000overview.aspx NanoDrop webpage] for more information.
 +
<br>
 +
<br>
 +
 
 +
===Tecan Infinite M200 Pro™===
 +
 
 +
For performing fluorescence measurements we used a Tecan Infinite M200 ProTM equipped with the Tecan iControl TM software.
 +
The Infinite 200 PRO is a user-friendly and affordable multimode reader, designed to cater for the needs of today’s applications. Based on the highly successful Infinite 200 series of microplate reader, the Infinite 200 PRO can provide a full range of leading detection methods in one easy-to-use modular instrument, with either Quad4 monochromator™ or filter-based technologies. Users can select the desired modules to create the perfect reader for their needs, with the option to upgrade as requirements change. The Infinite 200 PRO offers excellent sensitivity, multiplexing capabilities and high format flexibility, including 6- to 384-well microplates, PCR plates, cuvettes and Tecan’s patentedNanoQuant Plate™ for low sample volumes.
 +
<br>
 +
*For uncompromised performance in all detection modes, the Infinite 200 PRO uses advanced optics and high-performance detectors, optimized for the requirements of fluorescence, luminescence and absorbance reading. The Infinite 200 PRO offers a wide range of detection modes, including:
 +
*Fluorescence intensity (top and bottom reading) (UV – NIR & wavelength scanning)
 +
*Fluorescence resonance energy transfer (FRET)
 +
*Time resolved fluorescence energy transfer (TR-FRET) like HTRF®
 +
*Time resolved fluorescence (TRF)
 +
*Fluorescence polarization (FP)
 +
*Flash luminescence
 +
*Glow luminescence
 +
*Dual-color luminescence (including BRET 1 & BRET 2 applications)
 +
*Absorbance  (UV – NIR & wavelength scanning)
 +
*AlphaScreen® and AlphaLISA® technology
 +
*Injectors system for up to two reagents
 +
*Temperature control
 +
*Small sample measurement in NanoQuant Plate
 +
 
 +
[[File:ETH Zurich 2014 Tecan.jpg|center|500px|thumb|'''Figure 2'''Tecan Infinite M200 Pro™]]
 +
 
 +
Please visit the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan webpage] for more information.
 +
<br>
 +
<br>
 +
 
 +
===Tecan iControl™ software===
 +
Our Tecan is equipped with i-control, the easy-to-use microplate reader software. It allows the user to define the workflow for each application. Each workflow is easily created by dragging and dropping the processing steps into a sequence according to the assay protocol. The application workflow is then visible to the user and can be saved for future use. Data are easily managed and exported to Windows® compatible formats (Excel®).
 +
Intuitive
 +
Easy set up of workflows via drag & drop
 +
Workflows are visible to the user
 +
Workflows may be saved as measurement scripts
 +
On-line data presentation in Excel®
 +
Graphical definition of measurement range
 +
Plate definition editor
 +
Enhanced data management
 +
Export of data into user-defined Excel® templates
 +
Flexible
 +
User sets up the workflow to suit his own application
 +
Multilabel measurements for multiplexed assays
 +
Different kinds of kinetic measurements
 +
Supports ratio mode also for well kinetic measurements
 +
 
 +
Please visit the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1817&ID=1924&Menu=1&Item=21.7.8 Tecan software webpage] for more information.
 +
 
 +
 
 +
===Novaspec Plus™ Visible Spectrophotometer===
 +
 
 +
Novaspec™ Plus Visible Spectrophotometer is ideal for general biotech laboratory use. There are stored methods for the standard colorimetric methods of Bradford, BCA, Biuret and Lowry in addition to the basic modes of absorbance, transmittance,OD600 and concentration measurements. The use of diode array technology permits rapid wavelength scans to be made as well and, as there are no moving parts, make for a highly reliable and low maintenance product (Fig 1).
 +
The large backlit display enables the graphical results for wavelength scans, kinetics assays (including slope calculation for rate/activity studies) and standard curves to be viewed (Fig 2).Up to 99 methods for such experiments can be stored for instant recall and use by a laboratory assistant. Novaspec Plusis delivered with Grafico, a PC utility software package, and the requisite serial lead, providing the user with the means to capture, print and store data from the instrument (with a time/date stamp) onto a PC so that a results log can be built up or data exported to Microsoft™ Excel.
 +
 
 +
The cell holder supplied with Novaspec Plus accepts standard 10-mm pathlength glass or plastic cells (adapters are availableto convert it to accept 10-, 12- and 16-mm diameter test tubes).It can be removed for cleaning, or flushed through with water in situ, if spillages occur.
 +
For users who require thermostatting, particularly for kineticsstudies and use with test kits, a special version of Novaspec Plus is available with a factory fitted heated cell holder (37 °C only);note that this cell holder cannot be added retrospectively.
 +
 
 +
Features:
 +
 
 +
*“Flash Scan” diode array
 +
*Stored protein methods
 +
*Bacterial cell culture measurement at OD600
 +
*Kinetics for activity studies
 +
*Stored methods
 +
<br>
 +
Please visit the [http://www.heraco.se/images/user/PDF/Novaspec%20Plus.pdf Novaspec™ Plus Visible Spectrophotometer webpage] for more information.
 +
[[File:ETH Zurich 2014_Novaspec.jpg|center|400px|thumb|'''Figure 3''' Novaspec Plus™ Visible Spectrophotometer]]
 +
 
 +
===Biostep Dark-Hood DH-50™  and the Argus-X1™ software===
 +
<br>
 +
Pictures of our gels, long term imaging of our chips with beads containing GFP-expressing cells and pictures of our plates with bacterial colonies were recorded with the  DH-50™ and the Argus-X1™ software.
 +
 
 +
Dark Hood DH-50 with control panel, display, sliding table, UV protection, for CCD camera
 +
stationary dark hood with large door
 +
sliding table to put on a transilluminator incl. UV protection shield – no additional protection necessary
 +
dimmable white top-light by LEDs
 +
automatic UV cut-off when opening the UV protection shield
 +
preparative function enables observation of gels under UV light
 +
control panel with keypad and LCD display
 +
 
 +
Functionality:
 +
transilluminator: on/off
 +
white top-light: on/off, dim function
 +
preparative function
 +
optionally available: control of zoom, aperture and focus by motor zoom objective
 +
optionally available: storage of application methods repeatable
 +
* transilluminators with dimensions (W x D x H): 32.5x32.2x10.5cm; corresponds to the series of biostep UST, USDT, UXFT, BST, GST, YST, RST, WST
 +
optionally upgradeable with UV protection shield, filter wheel, white transmission light, UV or epi light in the visible range
 +
 
 +
 
 +
Please visit the [http://www.biostep.de/i18n_en/products/Bio_Imaging_components_1776/Tripod__dark_hoods_and_accessories_1863/Dark_hoods_1865/Dark_Hood_DH_50_6/index.html Biostep Dark-Hood DH-50™ webpage] for more information.
 +
 
 +
[[File:ETH Zurich 2014_Biostep Dark-Hood DH-50™.jpg|center|300px|thumb|'''Figure 4''' Biostep Dark-Hood DH-50™]]
 +
 
 +
===Biostep argusX1™ basic licence===
 +
 
 +
PC-software for controlling the camera basic modules Felix 1000/2000, 5000/6000/7000 and the transmission scanners of the series ViewPix 900/1300
 +
 
 +
Attention: Imperatively necessary: module for controlling the camera or scanner!!!
 +
 
 +
General software functions:
 +
integrated database for administration of the images
 +
copying and cropping of single images or group images within a database or even between database to database if the additional module Project administration exists
 +
deletion of single images or group images
 +
functions for image processing incl. Undo
 +
rotating in arbitrary angles and around 90°, 180°
 +
reflecting in horizontal and vertical direction
 +
inverting
 +
cropping
 +
various filter functions
 +
marking of the images with text, rectangle and line
 +
automated generation and numeration of sample names after the acquisition
 +
memo function: input of a note for each image and printout of the same on a protocol
 +
history for documentation of the acquisition parameters for each image
 +
overexposure control
 +
data export into other Windows applications
 +
data import of images in different formats
 +
creation of individual print reports
 +
quick print
 +
selection of the export format, export path, print report for quick print
 +
selection of the user language: German, English, Spanish, French or Italian
 +
control of the functions a of biostep transilluminators with PC interface
 +
 
 +
Please visit the [http://www.biostep.de/products/Bio_Imaging_software_1843/Acquisition_and_control_software_1844/argus_X1___for_cameras_and_scanners_1845/biostep_argusX1__basic_licence_1/index.html Biostep argusX1™ basic licence webpage] for more information.
 +
<br>
 +
<br>
 +
===Eppendorf Centrifuge 5810 R===
 +
 
 +
Centrifuge 5810/5810 R is a workhorse for medium to high-throughput laboratories. It combines extraordinary versatility and capacity for both tubes and plates with an extraordinary compact footprint. With the new rotor generation the capacity of Centrifuge 5810/ 5810 R increases to max. 4 x 750 mL (or max. 28 x 50 mL / 56 x 15 mL). Considering its small footprint, Centrifuge 5810/5810 R is the most compact 3 liter centrifuge currently available in the market.
 +
 
 +
Features:
 +
Swing-bucket rotors and adapters accommodate tubes and bottles from 0.2 mL to 750 mL
 +
Plate rotors for centrifugation of all types of MTP, PCR, cell culture, or deepwell plates
 +
Fixed-angle rotors for high-speed molecular biology applications in tubes from 0.2 mL to 85 mL
 +
High centrifugation speed of up to 20,913 x g (14,000 rpm)
 +
Aerosol-tight Eppendorf QuickLock® caps and lids for easy, one-hand operation
 +
Centrifuge lid with soft-touch lid closure
 +
Low access height of 29 cm for easy loading and unloading of samples
 +
Quiet operation to improve your work environment
 +
Compact footprint saves valuable bench space
 +
Automatic rotor recognition and imbalance detection for maximum operational safety
 +
Additional features of refrigerated Centrifuge 5810 R:
 +
Temperature range from -9 °C to 40 °C
 +
FastTemp function for fast pre-cooling
 +
Standby cooling function holds temperature when centrifuge is not in use
 +
ECO shut-off engages after 8 hours of non-use to reduce energy consumption and to extend compressor life
 +
Dynamic compressor control (DCC) technology for optimized cooling performance
 +
 
 +
Please visit the [http://www.eppendorf.com/int/index.php?pb=5ffa644d89305ae7&action=products&contentid=1&catalognode=10019&productpage=1 Eppendorf Centrifuge 5810 R webpage] for more information.
 +
 
 +
[[File:ETH Zurich 2014_Eppendorf Centrifuge 5810 R.jpg|center|300px|thumb|'''Figure 5''' Eppendorf Centrifuge 5810 R]]
 +
 
 +
===Eppendorf Centrifuge 5424 R===
 +
<br>
 +
This highly advanced 24-place model is unbelievable quiet and equipped with a number of innovative features. The Centrifuge 5424 and Centrifuge 5424 R set new standards for silence, speed and simplicity – for others to follow! The micro centrifuge standard in your lab – refrigerated. It features all the benefits already combined in the Centrifuge 5424 and adds the patented cooling feature for temperature sensitive samples.
 +
 
 +
Product features of Centrifuge 5424 and 5424 R
 +
Very compact size
 +
Very silent operation, even without the rotor lid
 +
Low access height – for easy loading and unloading of the centrifuge
 +
Can be equipped with four different rotors:
 +
- Standard 24-place rotor, aerosol-tight
 +
- 24-place rotor, aerosol-tight, PTFE coated for increased chemical resistance
 +
- 18-place Kit rotor for processing spin-columns with open tube lids
 +
- 4 × 8 PCR strip rotor
 +
Two different operation panel
 +
Rotary knobs for fast and easy setting of parameters
 +
Foil version for easier cleaning of the front panel
 +
Automatic lid opening following centrifuge run – prevents sample warming (can be optionally deactivated (only 5424))
 +
Separate Short-spin button – for fast and short spins
 +
SOFT-brake to protect delicate samples
 +
 
 +
 
 +
Additional cooling features of refrigerated Centrifuge 5424 R
 +
Patented compressor technology minimizes vibrations – protects your precious sample!
 +
Temperature range: –9 °C to +40 °C
 +
Small footprint and low access height of 26 cm, as refrigeration unit is at the rear of the unit
 +
Maintains constant 4 °C at max. speed
 +
Fast Temp for rapid pre-cooling of the centrifuge within 8 minutes from ambient temperature 23 °C
 +
Continuous cooling maintains temperature when centrifugation process is not active
 +
 
 +
 
 +
Please visit the [http://www.eppendorf.com/int/index.php?sitemap=2.1&pb=92b8536165b27ad1&action=products&contentid=1&catalognode=22420&productpage=1 Eppendorf Centrifuge 5424 R webpage] for more information.
 +
[[File:ETH Zurich 2014 Eppendorf Centrifuge 5424 R.jpg|center|300px|thumb|'''Figure 6''' Eppendorf Centrifuge 5424 R]]
 +
 
 +
===Harvard Pump 11 Plus===
 +
Our team used the syringe pump for fabricating the alginate beads containing our bacteria.
 +
 
 +
Harvard Apparatus’ Syringe Pump 11 Plus combines smoother flow and updated features to create a high performance syringe pump with a basic syringe pump price!
 +
 
 +
Bright Display and Easy-To-Use Interface
 +
A new, two-line 16 character vacuum fluorescent display and six membrane keys make this a powerful, easy-to-use syringe pump. Only two entries are required to start pumping; Syringe ID and flow rate. The flow rate can be changed while the pump is running.
 +
 
 +
Two Modes of Operation,
 +
Constant Flow Rate & Volume Dispense
 +
The new Pump 11 Plus will operate continuously in RATE mode or accurately dispense a specific amount of fluid in VOLUME mode.
 +
 
 +
Versatile
 +
Select from two different models. The standard Pump 11 Plus model has the following features:
 +
• Infusion Only
 +
• End of Travel Limit Stop
 +
• Anti-Siphon Bracket
 +
 
 +
Non-Volatile Memory
 +
New micro-stepping pump profiles deliver very smooth and consistent flow over the entire flow rate range. These pumps now feature non-volatile memory. The pump remembers its last syringe size, flow rate used and configuration settings. An advanced universal power supply means there is no need to change AC line switches, fuses or wires.
 +
 
 +
 
 +
Please visit the [https://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_44010_-1_HAI_ProductDetail___ Harvard Pump 11 Plus webpage] for more information.
 +
 
 +
[[File:ETH Zurich 2014_Harvard Apparatus syringe Pump 11 Plus.jpg|center|400px|thumb|'''Figure 7''' Harvard Apparatus syringe Pump 11 Plus]]
 +
 
 +
===BD LSRFortessa™ Flow Cytometer System===
 +
<br>
 +
In order to determine the exact sensivity shift and expression level of GFP with the mutated promoters the BD LSRFortessa™ Flow Cytometer System was used for the measurement,FACSDiva™ for the recording of the data and the FlowJo™ software for the data analysis.
 +
 
 +
BD LSRFortessa™ Flow Cytometer System
 +
 
 +
The BD LSRFortessa™ cell analyzer offers the ultimate in choice for flow cytometry, providing power, performance, and consistency.
 +
Designed to be affordable and expandable, the BD LSRFortessa has the flexibility to support the expanding needs of multicolor flow cytometry assays.The BD LSRFortessa analyzer can be configured with up to 4 lasers— blue, red, violet, and UV—which enables the detection of up to 18 colors simultaneously. The BD LSRFortessa may be upgraded subsequently with additional or new lasers from BD, as future requirements dictate.Innovative Designs for Optical Efficiency Built on a proven platform, BD LSR analyzers feature innovative designs for both the excitation optics and collection optics that reduce excitation losses and improve light collection efficiency. The result is optical efficiency that delivers maximum sensitivity and resolution for multicolor applications by reducing excitation laser light loss, and improved fluorescent signal collection.Special Order Research ProgramFor research needs at the leading edge of biomedical discovery, the BD LSRFortessa special order research program offers the latest innovations in laser technology to researchers to enable future upgrades with new excitation, wavelength, and power choices. The program lets customers fully customize configurations to deliver added flexibility and power to support advanced research.
 +
 
 +
 
 +
Please visit the [http://www.bdbiosciences.com/documents/bd_lsrfortessa_brochure.pdf BD LSRFortessa™ Flow Cytometer System webpage] for more information.
 +
[[File:ETH Zurich 2014_BD LSRFortessa™ Flow Cytometer System.jpg|center|400px|thumb|'''Figure 8''' BD LSRFortessa™ Flow Cytometer System]]
 +
 
 +
===FACSDiva™ software===
 +
BD FACSDiva™ software is a collection of rich tools for flow cytometer and application setup, data acquisition, and data analysis that help streamline flow cytometry workflows for today’s busy laboratory.
 +
 
 +
BD FACSDiva software provides new features to help users integrate flow systems into new application areas, including index sorting for stem cell and single-cell applications, as well as automation protocols for high-throughput and robotic laboratories.
 +
 
 +
Please visit the [http://www.bdbiosciences.com/instruments/software/facsdiva/ FACSDiva™ software webpage] for more information.
 +
 
 +
===MakerBot 3D-printer===
 +
 
 +
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot with MakerWare software; MakerBotIndustries, Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene (ABS, about US$160 per kg). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively.
 +
 
 +
[[File:ETH Zurich MakerBot.jpg|center|400px|thumb|'''Figure 9''' 2nd generation MakerBot]]
 +
 
 +
<html></article></html>
 +
 
 +
<html><article id='methods'></html>
 +
 
 +
==Methods==
 +
 
 +
===LB medium from dehydrated product===
 +
Suspend/dissolve the compounds in 1 L of purified water:
 +
 
 +
{| border="0"
 +
|-
 +
| align="left"  width="300"| Difco™ LB Agar, Miller || align="left" | 40 g
 +
|-
 +
| align="left" | Difco™ LB Broth, Miller || align="left" | 25 g
 +
|-
 +
| align="left" | ''Mix thoroughly'' ||
 +
|-
 +
| align="left" | ''Boil the agar medium with agitation for 1 min to dissolve the powder'' ||
 +
|-
 +
| align="left" | ''Autoclave at 121°C for 15 min'' ||
 +
|}
 +
 
 +
According to [https://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/244510.pdf LB Agar, Miller; LB Broth, Miller]
 +
<br>
 +
<br>
===Preparation of antibiotics stock (1000x)===
===Preparation of antibiotics stock (1000x)===
 +
{| border="0"
 +
|-
 +
| align="left" | Ampicillin (200 mg/mL) in H<sub>2</sub>O, sterile filtered
 +
|-
 +
| align="left"  | Kanamycin (50 mg/mL) in H<sub>2</sub>O, sterile filtered
 +
|-
 +
| align="left"  | Chloramphenicol (34 mg/mL) in ethanol
 +
|-
 +
| align="left"  | Tetracycline (10 mg/mL) in ethanol
 +
|-
 +
| align="left"  | Streptamycin (25 mg/mL) in H<sub>2</sub>O, sterile filtered
 +
|}
-
*Ampicillin (200 mg/mL) in H<sub>2</sub>O, sterile filtered
+
===Defined M9 medium with 0.4 % glycerol===
-
*Kanamycin (50 mg/mL) in H<sub>2</sub>O, sterile filtered
+
-
*Chloramphenicol (34 mg/mL) in ethanol
+
-
*Tetracycline (10 mg/mL) in ethanol
+
-
*Streptamycin (25 mg/mL) in H<sub>2</sub>O, sterile filtered
+
-
===SOC===
 
 +
Suspend/dissolve the sterile compounds in purified water to give 1 L final volume:
 +
{| border="0"
 +
|-
 +
| align="left"  width="300"| [http://openwetware.org/wiki/M9_salts M9 salts, 5x] || align="left" | 200 mL
 +
|-
 +
| align="left" | 30% (v/v) Glycerol || align="left" | 13.33 mL
 +
|-
 +
| align="left" | 10% (w/v) CAA || align="left" | 100 mL
 +
|-
 +
| align="left" | Thiamin (10 g/L) || align="left" |  2 mL
 +
|-
 +
| align="left" | TE-US solution<sup>[[Team:ETH_Zurich/project/references#refPanke|[33]]]</sup> || align="left" | 1 mL
 +
|-
 +
| align="left" | 1M CaCl<sub>2</sub> || align="left" | 0.5 mL
 +
|-
 +
| align="left" | 1M MgSO<sub>4</sub> || align="left" | 2 mL
 +
|}
 +
 +
<br/>
 +
<br/>
 +
 +
===SOC===
 +
 +
 +
Suspend/dissolve the compounds in 1 L of purified water:
{| border="0"  
{| border="0"  
Line 36: Line 345:
| align="left" | ''Autoclave, then add'' ||  
| align="left" | ''Autoclave, then add'' ||  
|-
|-
-
| align="left" | sterile glucose (1 M) || align="left" | 20 mL
+
| align="left" | Sterile glucose (1 M) || align="left" | 20 mL
|}
|}
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
 +
<br/>
 +
<br/>
===Transformation buffer 1 (TFB1)===
===Transformation buffer 1 (TFB1)===
 +
 +
Suspend/dissolve the compounds in 1 L of purified water:
{| border="0"  
{| border="0"  
Line 62: Line 375:
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
 +
<br/>
 +
<br/>
===Transformation buffer 2 (TFB2)===
===Transformation buffer 2 (TFB2)===
 +
Suspend/dissolve the compounds in 1 L of purified water:
{| border="0"  
{| border="0"  
Line 82: Line 398:
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
 +
<br/>
 +
<br/>
===Gibson Assembly reaction mixture===
===Gibson Assembly reaction mixture===
-
 
+
<h3>5x isothermal reaction buffer</h3>
-
 
+
-
<font size="5.5">5x isothermal reaction buffer</font>
+
{| border="0"  
{| border="0"  
|-
|-
Line 112: Line 428:
Aliquots can be stored at -20 °C
Aliquots can be stored at -20 °C
-
<font size="5.5">Assembly master mixture</font>
+
 
 +
 
 +
<h3>Assembly master mixture</h3>
{| border="0"  
{| border="0"  
|-
|-
Line 127: Line 445:
Aliquots of 15 μL can be stored at -20 °C
Aliquots of 15 μL can be stored at -20 °C
 +
<br/>
 +
<br/>
-
==Methods==
+
===Complex bead medium (CB medium)===
 +
 
 +
 
 +
{| border="0"
 +
|-
 +
| align="left"  width="300"| Yeast extract  || align="left" | 4 g
 +
|-
 +
| align="left" | Typtone || align="left" | 1 g
 +
|-
 +
| align="left" | Glycerol || align="left" | 1 g
 +
|-
 +
| align="left" | Tris(hydroxymethyl)aminomethane || align="left" | 1.21 g
 +
|-
 +
| align="left" | ''Fill up to 980 ml with H<sub>2</sub>O'' ||
 +
|-
 +
| align="left" | ''Adjust pH to 7  with HCl'' ||
 +
|-
 +
| align="left" | CaCl<sub>2</sub>&sdot;H<sub>2</sub>O (100 mM) || align="left" | 20 mL
 +
|-
 +
| align="left" | ''Sterilize by filtration'' ||
 +
|}
 +
 
 +
<br/>
 +
<br/>
-
===Preparation of competent ''E. coli''===
+
===Preparation of chemically competent ''E. coli''===
   
   
Day before: Preparation of a Top10 cells preculture  in LB-medium containing streptomycin (25 μg/mL)
Day before: Preparation of a Top10 cells preculture  in LB-medium containing streptomycin (25 μg/mL)
Line 145: Line 488:
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
 +
<br/>
 +
<br/>
 +
 +
===Preparation of electrocompetent ''E. coli''===
 +
# Grow a 5mL suspension of your cells of interest to OD=0.6
 +
# Pre-cool your centrifuge to 4°C
 +
# Pre-cool autoclaved water on ice
 +
# Cool the cells on ice for 5-10 minutes
 +
# Centrifuge for 6 minutes
 +
# Discard supernatant
 +
# Resuspend pellet with 1mL of chilled water
 +
# Centrifuge for 6 min
 +
# Repeat washing step followed by centrifugation 3 times
 +
# After last centrifugation step, discard supernatant and resuspend pellet in 50uL chilled water.
 +
# Add plasmid you want to transform
 +
# Electroporate
 +
# Add SOC medium and let cells recover at 37°C for 1 hour
 +
# Streak on LB-Agar plates with the corresponding antibiotic
===Preparation of DNA from iGEM kit===
===Preparation of DNA from iGEM kit===
Line 151: Line 512:
#Use 2 μL to transform competent cells
#Use 2 μL to transform competent cells
-
<font size="5">Biological parts used from iGEM kit:</font>
+
<h3>Biological parts used from iGEM kit:</h3>
{| border="0"  
{| border="0"  
|-
|-
Line 171: Line 532:
|}
|}
For confirmation all plasmids used from the iGEM kit were sequenced
For confirmation all plasmids used from the iGEM kit were sequenced
 +
<br/>
 +
<br/>
===Transformation of competent ''E. coli''===
===Transformation of competent ''E. coli''===
Line 184: Line 547:
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
 +
<br/>
 +
<br/>
===Plasmid preparation===
===Plasmid preparation===
Line 205: Line 570:
The [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html Sigma-Aldrich GenElute&trade; Plasmid Miniprep Kit] was used
The [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html Sigma-Aldrich GenElute&trade; Plasmid Miniprep Kit] was used
 +
<br/>
 +
<br/>
===Preparation of samples for sequencing at Microsynth===
===Preparation of samples for sequencing at Microsynth===
Add 12 μL DNA (60-100 ng/μL) to 3 μL of the corresponding primer (10 μM).
Add 12 μL DNA (60-100 ng/μL) to 3 μL of the corresponding primer (10 μM).
 +
<br/>
 +
<br/>
-
 
+
===PCR protocol for phusion DNA polymerase===
-
===PCR procol for phusion DNA polymerase===
+
{| class="wikitable"
{| class="wikitable"
Line 233: Line 601:
| H<sub>2</sub>O || add to reach a total volume of 20 μL ||  add to reach a total volume of 50 μL
| H<sub>2</sub>O || add to reach a total volume of 20 μL ||  add to reach a total volume of 50 μL
|}
|}
 +
<br/>
 +
<br/>
===Restriction Endonuclease Reaction (double digestion)===
===Restriction Endonuclease Reaction (double digestion)===
Line 251: Line 621:
Enzymes, buffers and protocol are from New England BioLabs
Enzymes, buffers and protocol are from New England BioLabs
 +
<br/>
 +
<br/>
===Dephosphorylation of 5’-ends of DNA using CIP===
===Dephosphorylation of 5’-ends of DNA using CIP===
Line 258: Line 630:
Enzymes, buffers and protocol are from New England BioLabs
Enzymes, buffers and protocol are from New England BioLabs
-
==DNA Purification by Centrifugation==
+
<br/>
-
<font size="5">A. Dissolving the Gel Slice</font>
+
<br/>
 +
 
 +
===DNA Purification by Centrifugation===
 +
<h3>A. Dissolving the Gel Slice</h3>
Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 ml microcentrifuge tube.
Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 ml microcentrifuge tube.
Line 266: Line 641:
or
or
-
<font size="5">B. Processing PCR Amplifications</font>
+
<h3>B. Processing PCR Amplifications</h3>
Add an equal volume of Membrane Binding Solution to the PCR mix.
Add an equal volume of Membrane Binding Solution to the PCR mix.
Line 279: Line 654:
#Add 50 µL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 min. Centrifuge at 16 000 rcf for 1 min.
#Add 50 µL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 min. Centrifuge at 16 000 rcf for 1 min.
#Discard Minicolumn and store DNA at 4 °C or –20 °C.
#Discard Minicolumn and store DNA at 4 °C or –20 °C.
-
 
According to Promega Wizard SV Gel and PCR Clean-Up System, Quick protocol
According to Promega Wizard SV Gel and PCR Clean-Up System, Quick protocol
 +
<br/>
 +
<br/>
===Agarose gel electrophoresis===
===Agarose gel electrophoresis===
-
 
#For a 5 cm x 6 cm gel add 0.25 g Agarose to 25 mL TAE (0.5x) and heat up in microwave to dissolve it
#For a 5 cm x 6 cm gel add 0.25 g Agarose to 25 mL TAE (0.5x) and heat up in microwave to dissolve it
Line 293: Line 668:
#Fill the samples and ladder in the wells
#Fill the samples and ladder in the wells
#Run the gel at 135 V
#Run the gel at 135 V
 +
<br/>
===Preparation of cryostocks===
===Preparation of cryostocks===
Line 298: Line 674:
#Add 750 μL sterile glycerol (30%) to 750 μL bacteria culture in a screw top tube
#Add 750 μL sterile glycerol (30%) to 750 μL bacteria culture in a screw top tube
#Freeze the glycerol stock tube at -80 °C
#Freeze the glycerol stock tube at -80 °C
 +
<br/>
===Site-directed mutagenesis===
===Site-directed mutagenesis===
Line 323: Line 700:
Protocol based on [http://2014.igem.org/wiki/index.php?title=Team:ETH_Zurich/lab/protocols&action=edit&section=19 QuikChange Site-Directed Mutagenesis]
Protocol based on [http://2014.igem.org/wiki/index.php?title=Team:ETH_Zurich/lab/protocols&action=edit&section=19 QuikChange Site-Directed Mutagenesis]
 +
<br/>
 +
<br/>
===Gibson Assembly===
===Gibson Assembly===
Line 333: Line 712:
# Run the reaction for 30-60 min at 50 °C
# Run the reaction for 30-60 min at 50 °C
# Subsequently the reaction mixture (5 uL are enough) can be used directly to transform competent cells (75 uL)
# Subsequently the reaction mixture (5 uL are enough) can be used directly to transform competent cells (75 uL)
 +
<br/>
 +
 +
 +
===Multi site directed mutagenesis===
 +
 +
Single Primer Method
 +
 +
This protocol only uses one oligo per mutation site (cuts the oligo cost in half) and can mutate more than one site at a time. While this protocol is less reliable than the previous protocol (only ~25% of colonies will contain the desired mutation(s)), if you're doing multiple mutations on the same piece of DNA than this can save you many days of work by just sequencing a few more colonies. In this protocol there is an initial Kinase (phosphorylation) reaction to allow your primers to be available for ligation. The polymerase will extend from one primer to another and then the Taq ligase will seal the nick. This allows multiple mutations to be done at the same time.
 +
 +
Method
 +
#Design mutagenesis primer(s). The targeted mutation should be in the middle of the primer. Design your primers (including the mutations) to have a Tm >=78°C.
 +
#Purify template plasmid from a dam+ ''E. coli'' strain via miniprep.
 +
#Set up mutagenesis PCR mix:
 +
<br>
 +
16 μL Water
 +
<br>
 +
1.5 μL DMSO(100%)
 +
<br>
 +
0.5 μL MgSO4(50 µM)
 +
<br>
 +
10 μL Phusion HF Buffer
 +
<br>
 +
5 μL T4 Ligase Buffer
 +
<br>
 +
5 μL 10x Taq Ligase Buffer
 +
<br>
 +
0.5 μL Each Mutagenesis Primer (at 40 µM)
 +
<br>
 +
2 μL Reverse Biobrick Primer
 +
<br>
 +
2 μL dNTPs (100mM)
 +
<br>
 +
2 μL Template
 +
<br>
 +
2 μL PNK
 +
<br>
 +
2 μL Taq Ligase
 +
<br>
 +
1 μL Phusion Polymerase (hotstart)
 +
<br>
 +
#Run PCR
 +
*37°C for 30 minutes (this is the kinase step)
 +
*95°C for 3 minutes
 +
*Run the following for 20 cycles:
 +
**95°C for 1 minute
 +
**55°C for 1 minute
 +
**65°C for 30 sec/kb of plasmid length minimum
 +
**4°C infinite
 +
#Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary)
 +
#Incubate 4-6 hours at 37°C.
 +
#Purify PCR product and elute into 30μL.
 +
#Transform 3μL purified DNA into highly competent cells.
 +
#Screen the transformants for the desired mutation using restriction digest or sequencing.
 +
<br>
 +
Notes
 +
Because the inital kinase step could lead to non-specific binding, it would be best to use a hot-start polymerase for this procedure.
 +
Any combination of DMSO and MgSO4 has shown significant improvments in product.
 +
 +
According to [http://openwetware.org/wiki/Richard_Lab:Site_Directed_Mutagenesis Multi Site Directed Mutagenesis, Single Primer Method]
 +
 +
<br>
 +
<br>
===Preparation of alginate beads===
===Preparation of alginate beads===
Line 342: Line 783:
#Add bacteria-NaCl-suspension to sterile alginate (2.5%) so as to reach an alginate concentration of 2%
#Add bacteria-NaCl-suspension to sterile alginate (2.5%) so as to reach an alginate concentration of 2%
#Fill the suspension into a sterile syringe  
#Fill the suspension into a sterile syringe  
-
 
+
#Use a suitable device for uniform droplet formation
-
blablabla
+
#Collect the droplets in a beaker containing CaCl<sub>2</sub> (100mM) to allow electrostatic cross-linking within the alginate bead
-
 
+
#Store the beads in CaCl<sub>2</sub> (10mM)
-
1 bead: approximatly 14.5 µl alginate-NaCl
+
1 bead corresponds to approximatly 14.5 µl alginate-NaCl
-
{{:Team:ETH_Zurich/tpl/foot}}
+
<html></article></html>

Latest revision as of 23:45, 17 October 2014

iGEM ETH Zurich 2014