Revision as of 13:30, 10 October 2014

Analytical methods

Agarose gel electrophoresis

Separation of DNA or RNA

take 5µl of the PCR product
mix with 1µl loading dye
apply onto agarose gel together with a marker
run at 120°C for 40 minutes for a full gel

SDS-PAGE

Cell preparation

lysis cell pellet in lysis buffer
centrifuge for 15 min at 13.000 rpm
mix the supernatant with 2x lammli buffer with β-mercaptoethanol
denatured for 5 min at 95 °C
sample to the gel

For some SDS-PAGEs, we used BioRad ready made gels.

The recipe of the self-made SDS is as follows:

1.5x Buffer

1.5 M Tris-Cl pH = 8.8
in 1 L is 40 ml 10 % SDS

Gels

	0.75 mm 12 % RUNNING Gel			1 mm 4 % STACKING Gel
	1x	2x	4x	1x	2x	4x
H₂O	1.65 mL	3.3 mL	6.6 mL	1.5 mL	3 mL	6 mL
1.5x Gel Buffer	1.3 mL	2.6 mL	5.2 mL	0.65 mL	1.3 mL	2.6 mL
30 % Acrylamide (37.5:1)	2 mL	4 mL	8 mL	0.325 mL	0.65 mL	1.3 mL
10 % APS	50 µL	100 µL	200 µL	25 µL	50 µL	100 µL
TEMED	10 µL	20 µL	40 µL	5 µL	10 µL	20 µL

Run gel

apply the prepared samples together with a protein marker on the gel
run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V

Bradford assay

Determination of protein concentration

Measurement of fluorescence

The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.

volume of sample in each well: 100µl
measure GFP fluorescence at an excitation wavelength of 496 nm and an emission wavelength at 516 nm

Measurement of optical density

Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.

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@@ Line 4: / Line 4: @@
 {{Team:Aachen/Header}}
 <span id="partners"></span>
-= Molecular biological methods =
-== Cloning ==
-=== Restriction Digest ===
-=== Ligation ===
-=== Gibson Assembly ===
-The Gibson Assembly was conducted according to the protocol published by [https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510 New England Biolabs].
-# Set up the reaction according to the table below on ice (2-3 fragment assembly).
-# Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
-# Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
-<center>
-{| class="wikitable" style="text-align: right;"
-|-
-| '''Total Amount of Fragments''' || 0.02-0.5&nbsp;pmols
-|-
-| '''Gibson Assembly Master Mix (2X)''' || 10&nbsp;µl
-|-
-| '''Deionized H<sub>2</sub>O''' || 10-X&nbsp;µl
-|-
-| '''Total Volume''' || '''20&nbsp;µl'''
-|-
-|}
-</center>
-== Transformation ==
-=== Heat Shock ===
-# thaw cells on ice
-# add 1&nbsp;µL of plasmid DNA
-# incubate on ice for 30 min
-# heat shock at 42&nbsp;°C for 60 s
-# incubate on ice for 5 min
-# add 200&nbsp;µL of SOC media
-# incubate at 37&nbsp;°C for 2&nbsp;h
-# plate 20&nbsp; and 200&nbsp;µL on plates supplemented with the appropiate antibiotic
-=== Electroporation ===
-# add 1&nbsp;μL plasmid to electrocompetent cells
-# put DNA/ cell suspension in electroporation cuvette
-# wipe dry the electroporator
-# use a small plastic pipette to place the cells
-# pulse: 2.5&nbsp;kV, 200-400&nbsp;Ω, 25&nbsp;μF (for ''E.coli'')
-# immediatly add 1&nbsp;mL LB and incubate for 2&nbsp;h at 37&nbsp;°C
-# plate 50&nbsp;μL on selective medium plate
-# centrifuge the rest (3000&nbsp;g, 20 min), discard supernatant, re-suspend the pellet in 50&nbsp;μL LB and plate it on selective medium plate
-== PCR ==
-We have used several different types of PCR throughout our project:
-* colony PCR
-* check PCR
-* gradient PCR
-* SOE PCR
-* touchdown PCR
-* QuikChange(Ligation-During-Amplification)
-The scope of appplication as well as the conduct are described below.
-=== Colony PCR /Check PCR ===
-'''With GoTaq Mast Mix'''
-* 12.5 µl GoTaq Master Mix
-* 1 µl primer_F
-* 1 µl primer_R
-* pick colony with tip and suspend in PCR tube
-* 9.5 µl ddH<sub>2</sub>O
-<center>
-{| class="wikitable"
-! parameter !! duration !! temp [°C] !!
-|-
-| denature||5:00||95 ||
-|-
-| '''anneal'''||00:30||56 || rowspan="3" | 30 cycles
-|-
-| '''elongate'''||01:00 per kb||72
-|-
-| '''denature'''||00:30||95
-|-
-| elongate||05:00||72 || rowspan="2" |
-|-
-| store||forever||8
-|}
-</center>
-=== gradient PCR ===
-=== SOE PCR ===
-=== touchdown PCR ===
-=== QuikChange ===
-{{Team:Aachen/BlockSeparator}}
 = Analytical methods =

Team:Aachen/Notebook/Protocols/Analytical methods

From 2014.igem.org