Revision as of 12:59, 17 June 2017

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

Chemical Transformation

Materials:
LB broth (Luria Bertrani medium = rich media to grow bacteria)
TSS buffer (to prepare chemically competent cells)
S.O.C. medium (helps obtain the maximal transformation efficiency)
LB agar (gel where bacteria can grow)
Chloramphenicol (CAL) at stock concentration 25mg/ml

Preparation of chemical competent cells (see: TSS Competent E. coli Protocol):

Inoculate DH5α cells into 50mL LB and incubate at 37°C
Monitor growth every 30mins by measuring optical density at 600nm (OD600) until it reaches OD600 = 0.4-0.6
Harvest cells and prepare using TSS protocol (see: TSS Competent E. coli Protocol)
Aliquot 200μL and flash freeze at -80°C

LB Agar plates preparation:

Prepare LB containing chloramphenicol (CAL) (at 25μg/ml)

Melt LB in microwave (defrost setting for 15mins)
Cool LB by running cold water over
Stock of 25mg/ml CAL → so add 400μl CAL to 400ml LB

Pour plates (in fume hood) and allow to solidify

Chemical Transformation:

Add 1μl of DNA to 50μl of competent cells, mix well and place on ice for at least 30mins
Heat shock cells at 42°C for 30secs, followed by 2min incubation on ice
Add 450μl of SOC medium tot he cells and incubate for 45min at 37°C (to allow (antibiotic resistance) protein expression)
Plate and spread (glass spreader sterilised over a flame and in ethanol) 50, 100, and 200μl of the cells into the agar plates made previously
Incubate at 37°C for 2h

TSS Competent E. coli Preparation

Materials:
Dimethyl sulfoxide (DMSO)
Polyethylene glycol (PEG)
MgCl2 stock solution
LB or SOC liquid medium
Ice

Methods:

Inoculate DH5α E. coli cells into 50ml LB broth and incubate at 37°C
Prepare the TSS buffer while waiting for the culture to grow and place it on ice (see: Table below)
Monitor growth of the culture every 30min by measuring the optical density at 600nm wavelength (OD600) until it reaches OD600 = 0.4-0.6 (takes approximately 2-3h)
Once the proper optical density has been achieved, take 100ml of culture and centrifuge under 2.700xg for 10 min at 4°C
Resuspend each tube in 5ml of pre-chilled TSS buffer with gentle vortexing
Chill TSS suspended cells on ice for 15 min
Aliquot 200μL of TSS suspended cells while ensuring the cells remain well mixed
Cells can be used immediately or stored at -80°C

TSS Buffer Composition

Component	Stock (M)	Amount
MgCl2	2	0.300ml
DMSO	-	1ml
PEG (3350 or 8000)	-	2g
LB Medium	to final volume of 20ml	to final volume of 20ml

PCR From Plasmid DNA Template

Materials:
2x Q5 Master Mix
10 µM forward primer
10 µM reverse primer
DNA template
Nuclease-free water

Methods:

Gently mix the reaction from the components listed in the table below and place it on ice
Note: addition of reagents are done in the following order to prevent degradation of primers: nuclease-free water, both primers, DNA, and then Q5 mix

PCR Reaction Components

Component	25µl Reaction	50µl Reaction	Final concentration
Q5 High-Fidelity 2X Master Mix	12.5µl	25µl	1X
10µM Forward Primer	1.25µl	2.5µl	0.5µM
10µM Reverse Primer	1.25µl	2.5µl	0.5µM
Template DNA	variable	variable	< 1,000ng
Nuclease-Free Water	to 25µl	to 50µl

When necessary, collect all liquid to the bottom of the PCR tube by spinning for a short time
Transfer the PCR tube from ice to a PCR machine and begin thermocycling

Thermocycling
The PCR machine should be set to run the following steps:

Step	Temperature (°C)	Time
Initial denaturation	98	30 seconds
30 cycles	98 (denaturation) 63 (annealing) see Note 1 72 (extension)	5 seconds 30 seconds 27 seconds per kb
Final extension	72	2 minutes
Hold	10	-

Note 1: The NEB Tm calculator should be used to determine the annealing temperature when using the Q5 Master Mix: http://tmcalculator.neb.com/#!/

Gel Electrophoresis

Materials:
1% TAE Buffer
Agarose powder
SYBR Safe
Loading dye
DNA ladder

Make 1% agarose gel:

Prepare 1% TAE agarose gel: dissolve 1g of agarose into 100ml of TAE buffer in a conical flask
Warm in microwave for 1min at max power
Remove flask from microwave with care, swirl gently and cool under running tap
Add 5µl of SYBR Safe
Prepare a casting tray with suitable comb
Pour to cool mixture into the casting tray and wait 15min until it solidifies

Run gel:

Add 5µl of PCR solution and 1µl 10x loading dye
Load 6ul of DNA ladder alongside and all samples (Do not forget to add dye to ladder too) - NEB 1kb ladder used
Run gel at 100V for 45min
Visualise gel on a transilluminator (SYBR Safe binds DNA and fluoresces under UV light)

PCR Purification (QIAquick PCR Purification Kit)

Materials:
Ethanol (96-100%)
Buffer PE
Buffer PB
Buffer EB
pH indicator I
Sodium acetate
QIAquick column
Loading dye

Notes before starting:

Add ethanol (96-100%) to buffer PE before use (see bottle label for volume)
All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature
Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots

Methods:

Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10μl 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
Place a QIAquick column in a provided 2 ml collection tube
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 seconds until all the samples have passed through the column. Discard flow-through and place the QIAquick column back in the same tube
To wash, add 0.75ml Buffer PE to the QIAquick column and centrifuge for 30–60 seconds. Discard flow-through and place the QIAquick column back in the same tube
Centrifuge the QIAquick column once more in the provided 2ml collection tube for 1 min to remove residual wash buffer
Place each QIAquick column in a clean 1.5ml microcentrifuge tube
To elute DNA, add 50μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
If the purified DNA is to be analyzed on a gel, add 1 volume of Loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel

@@ Line 397: / Line 397: @@
 <li>Place each QIAquick column in a clean 1.5ml microcentrifuge tube</li>
 <li>To elute DNA, add 50μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge</li>
-<li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel</li>
+<li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel</li>
 </ol>
 </p>

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