Difference between revisions of "Team:Aachen/Notebook/Protocols"
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{{Team:Aachen/Protocol|title=B0034_Insertion-Mutagenesis|id=B0034_Insertion-Mutagenesis|text=<span style="display:none;">spacer</span>With this method B0034 can be inserted into any RFC10 part that begins with prefix-CDS. | {{Team:Aachen/Protocol|title=B0034_Insertion-Mutagenesis|id=B0034_Insertion-Mutagenesis|text=<span style="display:none;">spacer</span>With this method B0034 can be inserted into any RFC10 part that begins with prefix-CDS. | ||
− | The protocol is taken from Bryksin et al. 2014<ref>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.</ref>. | + | The protocol is taken from Bryksin et al. 2014<ref>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.</ref>. |
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{{Team:Aachen/Protocol|title=Nash Assay|id=Nash_Assay|text=<span style="display:none;">spacer</span> | {{Team:Aachen/Protocol|title=Nash Assay|id=Nash_Assay|text=<span style="display:none;">spacer</span> | ||
− | === | + | ===Principle of detection=== |
− | The colorimetric and fluorometric assay first described by T. Nash in 1953 <ref>Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. ''Biochemical Journal'', '''55'''(3), 416-421.</ref> | + | The colorimetric and fluorometric assay first described by T. Nash in 1953<ref>Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. ''Biochemical Journal'', '''55'''(3), 416-421.</ref> detects the presence of formaldehyde. Two molecules of acetylacetone react with formaldehyde and ammonia to form the strong yellow and fluorescing diacetyl-dihydro lutidine which can be detected via absorption at a wavelength of 412 nm and via fluorescence at an excitation wavelength of 410 nm and an emission wavelength of 510 nm. |
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* mix Nash reagent : reaction buffer : sample in the ratio of 20 : 19 : 1 in a MTP/cuvette | * mix Nash reagent : reaction buffer : sample in the ratio of 20 : 19 : 1 in a MTP/cuvette | ||
* Measurement | * Measurement | ||
− | ** measure | + | ** measure absorbance at 412 nm |
** measure fluorescence with an excitation wavelength of 410 nm and an emission wavelength of 510 nm | ** measure fluorescence with an excitation wavelength of 410 nm and an emission wavelength of 510 nm | ||
** measure hourly starting directly after mixing for at least 5 hours | ** measure hourly starting directly after mixing for at least 5 hours | ||
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{{Team:Aachen/Protocol|title=HPLC|id=HPLC|text=<span style="display:none;">spacer</span> | {{Team:Aachen/Protocol|title=HPLC|id=HPLC|text=<span style="display:none;">spacer</span> | ||
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− | + | *for methanol | |
− | + | <table class="wikitable"> | |
− | + | <tr> | |
− | + | <th>Retention time</th> | |
− | + | <th>Lower detection limit</th> | |
− | + | </tr> | |
+ | <tr> | ||
+ | <td>16,76 +/- 0,02 min</td> | ||
+ | <td>~20 mM</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | *for sugars | ||
+ | <table class="wikitable"> | ||
+ | <tr> | ||
+ | <th></th> | ||
+ | <th></th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>HPLC</td> | ||
+ | <td>Beckman System Gold</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Column</td> | ||
+ | <td>Organic Acid Resin (PS-DVB), 300 x 8.0 mm, CS-Chromatographie</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Detector</td> | ||
+ | <td>RI 166, Beckman</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Column Temperature</td> | ||
+ | <td>75°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Solvent</td> | ||
+ | <td>5 mM H2SO4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Flow Rate</td> | ||
+ | <td>0.8 ml/min</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Duration</td> | ||
+ | <td>20 min</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Injection Volume</td> | ||
+ | <td>10 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Tray Cooling</td> | ||
+ | <td>5°C</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | Samples have to be centrifuged in order to remove solids (cells/precipitates). No further sample preparation is required. | ||
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}} | }} | ||
− | {{Team:Aachen/Protocol|title=Dinitrosalicylic Acid Staining|id=Dinitrosalicylic_Acid_Staining |text=The | + | {{Team:Aachen/Protocol|title=Dinitrosalicylic Acid Staining|id=Dinitrosalicylic_Acid_Staining |text=The dinitrosalicylic acid assay is used to analyse the distribution of branches in Glycogen. Dinitrosalicylic acid reacts with the reducing ends of glycogen (or sugars). <ref>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). |
</ref> | </ref> | ||
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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 | + | # Take the purified samples (purification via [[Team:Aachen/Notebook/Protocols#Glycogen Kit|Glycogen Kit]] section '''Pre-extraction of glycogen''') and add one volume of dinitrosalicylic acid solution (solve 30 g dinitrosalicylic acid in 20 mL 2 M NaOH and add 80 mL water) |
− | # Heat stained | + | # Heat stained samples at 90 °C for 15 minutes at 800 rpm |
− | # Add one third of volume of | + | # Add one third of the total volume of potassium sodium tartarte solution (40 % wt/vol) to samples, to stabilyze the color |
# Cool samples down to 25 °C | # Cool samples down to 25 °C | ||
− | # Analyze samples at 540 nm | + | # Analyze samples at 540 nm absorbance and substract the absorbance value of color blank each time |
}} | }} | ||
Latest revision as of 03:37, 19 September 2015