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

 
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−
#  for plates, let it cool to '''60°C'''
+
#  for plates, let it cool to '''60 °C'''
 
#  add antibiotics and mix thoroughly
 
#  add antibiotics and mix thoroughly
 
#  pour plates and pile
 
#  pour plates and pile
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'''Endconcentrations'''
 
'''Endconcentrations'''
  
−
*MgSO4 (1 mM)
+
*MgSO{{sub|4}} (1 mM)
 
*Traceelement Solution
 
*Traceelement Solution
 
*Glucose (80 mM)
 
*Glucose (80 mM)
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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&nbsp;et&nbsp;al.&nbsp;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&nbsp;et&nbsp;al.&nbsp;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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# Linearize the destination vector by amplification with suitable primers. Insert(s) must be amplificated with primers that contain overhangs to create overlapping regions with the backbone (and additional parts).
 
# Linearize the destination vector by amplification with suitable primers. Insert(s) must be amplificated with primers that contain overhangs to create overlapping regions with the backbone (and additional parts).
 
# Mix 100 ng of each fragment together with calculated amounts of H{{sub|2}}O, DMSO, dNTPs and DNA polymerase
 
# Mix 100 ng of each fragment together with calculated amounts of H{{sub|2}}O, DMSO, dNTPs and DNA polymerase
−
# Repeat 15 cycles of denaturation (98°C), annealing and elongation in a thermocycler
+
# Repeat 15 cycles of denaturation (98 °C), annealing and elongation in a thermocycler
 
# Transform the construct in a strain of your choice
 
# Transform the construct in a strain of your choice
  
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*Set up the reaction according to the table below on ice (2-3 fragment assembly).
 
*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.
+
*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.  
 
*Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.  
  
Line 253: Line 253:
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td>Deionized H2O</td>
+
<td>Deionized H{{sub|2}}O</td>
 
<td>10-X µl</td>
 
<td>10-X µl</td>
 
</tr>
 
</tr>
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'''Procedure'''
 
'''Procedure'''
−
# Resuspend cell pellet (or dried crudely extracted glycogen) in 500 µL 5M HCl in an appropriately sized glass vial
+
# Resuspend cell pellet (or dried crudely extracted glycogen) in 500 µL 5 M HCl in an appropriately sized glass vial
−
# Cook sample with tightly closed lid for at least 6 hours at 105°C
+
# Cook sample with tightly closed lid for at least 6 hours at 105 °C
−
# Dry sample until black/brown residue is left. This is done at 85°C with no lid under a hood with constant airflow!
+
# Dry sample until black/brown residue is left. This is done at 85 °C with no lid under a hood with constant airflow!
 
# Store samples at room temperature
 
# Store samples at room temperature
 
}}
 
}}
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Notes:
 
Notes:
 
* For samples having glucose background, prepare well(s) containing same amont of sample as in the test well as background control.
 
* For samples having glucose background, prepare well(s) containing same amont of sample as in the test well as background control.
−
* (Accurate determination of Glycogen in the test samples is best determined via spiking samples with a known amount of standard (example given 0.8 µg))
+
* (Accurate determination of Glycogen in the test samples is best determined via spiking samples with a known amount of standard (example given 0.8 µg)
  
  
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−
3. Add (1 µL) Hydrolysis Enzyme mix to Standards and samples and mix well.
+
3. Add 1 µL Hydrolysis Enzyme mix to Standards and samples and mix well.
  
  
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−
3. Add (1 µL?) Hydrolysis Enzyme mix to Standards and samples and mix well.
+
3. Add 1 µL Hydrolysis Enzyme mix to Standards and samples and mix well.
  
  
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# Heat to 100 °C for 2 h
 
# Heat to 100 °C for 2 h
 
# Cool and add 2 volumes of 95% ethanol to precipitate crude glycogen
 
# Cool and add 2 volumes of 95% ethanol to precipitate crude glycogen
−
# Centrifuge (16 000g for 10 min) and collect the precipitate
+
# Centrifuge (16000 g for 10 min) and collect the precipitate
 
# Suspend the pellet in a minimal amount of distilled water (25 ml original cell culture -> 5 ml were enough)
 
# Suspend the pellet in a minimal amount of distilled water (25 ml original cell culture -> 5 ml were enough)
−
# Acidify sample to pH3 with 5 M HCl
+
# Acidify sample to pH 3 with 5 M HCl
 
# Add 1 volume of ethanol to reprecipitate
 
# Add 1 volume of ethanol to reprecipitate
 
# Repeat steps 4-7 two more times, then let sample dry over night (pre-weigh container for drying)
 
# Repeat steps 4-7 two more times, then let sample dry over night (pre-weigh container for drying)
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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>
−
===Priciple of detection===
+
===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> detetcts 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 adsorption at a wavelength of 412 nm and via fluorescence at an excitation wavelength of 410 nm and an emission wavelength of 510 nm.
+
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 adsorption at 412 nm
+
** 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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# inoculate main culture (50 ml) with a start OD of 0.15
 
# inoculate main culture (50 ml) with a start OD of 0.15
 
# induce with IPTG at OD 0.6
 
# induce with IPTG at OD 0.6
−
# Take samples (2ml)
+
# Take samples (2 ml)
 
## sample: 5-6 hours after induction
 
## sample: 5-6 hours after induction
 
## sample: ca 18 hours after induction
 
## sample: ca 18 hours after induction
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<tr>
 
<tr>
 
<th>Ingredient</th>
 
<th>Ingredient</th>
−
<th>Stacking Gel (1mL) </th>
+
<th>Stacking Gel (1 mL) </th>
 
<th>Running Gel (5 mL; 15%)</th>
 
<th>Running Gel (5 mL; 15%)</th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td>H{{sub|2}}O</td>
 
<td>H{{sub|2}}O</td>
−
<td>0,72 mL</td>
+
<td>0.72 mL</td>
−
<td>1,73 mL </td>
+
<td>1.73 mL </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td>1,5 M Tris</td>
+
<td>1.5 M Tris</td>
−
<td>0,13 mL (pH 6,8)</td>
+
<td>0.13 mL (pH 6.8)</td>
−
<td>1,3 mL (pH 8,8)</td>
+
<td>1.3 mL (pH 8.8)</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td>10% Ammoniumperoxodisulfat</td>
+
<td>10 % Ammoniumperoxodisulfat</td>
−
<td>0,01 mL</td>
+
<td>0.01 mL</td>
−
<td>0,05 mL</td>
+
<td>0.05 mL</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td>10% N,N′-Methylenbisacrylamid</td>
+
<td>10 % N,N′-Methylenbisacrylamid</td>
−
<td>0,13 mL</td>
+
<td>0.13 mL</td>
−
<td>0,05 mL</td>
+
<td>0.05 mL</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td>30% Acrylamid</td>
+
<td>30 % Acrylamid</td>
−
<td>0,13 mL</td>
+
<td>0.13 mL</td>
−
<td>1,87 mL</td>
+
<td>1.87 mL</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td>TEMED</td>
 
<td>TEMED</td>
−
<td>0,001 mL</td>
+
<td>0.001 mL</td>
−
<td>0,002 mL</td>
+
<td>0.002 mL</td>
 
</tr>
 
</tr>
 
</table>
 
</table>
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}}
 
}}
  
−
{{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).
+
 
 +
{{Team:Aachen/Protocol|title=HPLC|id=HPLC|text=<span style="display:none;">spacer</span>
 +
 
 +
 
 +
*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.
 +
 
 +
 
 +
}}
 +
 
 +
{{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>
  
  
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−
# Take the purified samples (purification via [[Team:Aachen/Notebook/Protocols#Glycogen_Kit|Purification Protocol]]) 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 one 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 samples at 90 °C for 15 minutes at 800 rpm
−
# Add one third of volume of potassiumsodium tartarte solution (40 % wt/vol) to samples
+
# 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 adsorbance in plate reader
+
# Analyze samples at 540 nm absorbance and substract the absorbance value of color blank each time
 
}}
 
}}
 +
 +
  
 
<div class="col-md-12"><h1>References</h1></div>
 
<div class="col-md-12"><h1>References</h1></div>

Latest revision as of 03:37, 19 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[+]


HPLC[+]

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).


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