Difference between revisions of "Team:Aachen/Notebook/Protocols"

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# Take the purified samples (purification via [[Team:Aachen/Notebook/Protocols#Glycogen Kit|Glycogen Kit]] section '''Pre-extraction of glycogen''') and add the same volume of dinitrosalicylic acid solution (solve 30 g dinitrosalicylic acid in 20 mL 2 M NaOH and add 80 mL water).
 
# Take the purified samples (purification via [[Team:Aachen/Notebook/Protocols#Glycogen Kit|Glycogen Kit]] section '''Pre-extraction of glycogen''') and add the same volume of dinitrosalicylic acid solution (solve 30 g dinitrosalicylic acid in 20 mL 2 M NaOH and add 80 mL water).
# Heat stained sample at 90°C for 15 minutes.
+
# Heat stained sample at 90 °C for 15 minutes.
 
# Add one third of volume of potassiumsodium tartarte solution (40 % wt/vol) to samples
 
# Add one third of volume of potassiumsodium tartarte solution (40 % wt/vol) to samples
# Cool samples down to 25°C
+
# Cool samples down to 25 °C
 
# Analyze samples at 540 nm adsorbance in plate reader
 
# Analyze samples at 540 nm adsorbance in plate reader
 
}}
 
}}

Revision as of 20:10, 18 September 2015


The most important recipe for effective research and reproducible results are reliable protocols.

On this page we are providing several protocols that we have used over the course of our project.


Culturing


Lysogeny-Broth (LB Medium)[+]

M9 Medium[+]


Overnight Cultures[+]


SOC Medium[+]

Cloning


B0034_Insertion-Mutagenesis[+]

CPEC[+]


Gibson Assembly[+]


Genomic Amplification[+]

Transformation[+]

Plasmid Preparation[+]


RDP Assembly[+]

Analytics

Acid Hydrolysis[+]

Cell Lysis[+]


Electrophoresis[+]

Glycogen Kit[+]

Iodine Staining[+]

Nash Assay[+]


SDS-PAGE[+]

Dinitrosalicylic Acid Staining[+]

References

  1. Bryksin AV, Bachman HN, Cooper SW, Balavijayan T, Blackstone RM, Du H, Jenkins JP, Haynes CL, Siemer JL, Fiore VF, Barker TH. One primer to rule them all: universal primer that adds BBa_B0034 ribosomal binding site to any coding standard 10 BioBrick. ACS Synth Biol. 2014 Dec 19;3(12):956-9. doi:10.1021/sb500047r. PubMed PMID: 25524097; PubMed Central PMCID: PMC4277749.
  2. Quan, Jiayuan, and Jingdong Tian. “Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways.” Ed. Paulo Lee Ho. PLoS ONE 4.7 (2009): e6441. PMC. Web. 16 Sept. 2015.
  3. https://j5.jbei.org/j5manual/pages/22.html
  4. https://www.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly
  5. http://www.geneious.com
  6. http://parts.igem.org/Help:Transformation_Protocol Protocol of the iGEM HQ
  7. Blank Lars, Protocol for 13C Tracer Experiments
  8. [http://www.biovision.com/manuals/K646-100.pdf Glycogen Assay Kit Manual]
  9. https://www.thermofisher.com/de/de/home/references/ambion-tech-support/rna-tools-and-calculators/macromolecular-components-of-e.html
  10. http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp
  11. Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochemical Journal, 55(3), 416-421.
  12. Kleeberg & Klinger. 1982. Sensitive formaldehyde determination with Nash's reagent and a tryptophan reaction. Journal of Pharmacological Methods, 8(1), 19-31.
  13. S. K. Meur, V. Sitakara Rao, and K. B. De. Spectrophotometric Estimation of Reducing Sugars by Variation of pH. Z. Anal. Chem. 283, 195-197 (1977)