Difference between revisions of "Team:Aix-Marseille/Experiments/Protocols"

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== Protocol #0 : iGEM General Protocol ==
 
== Protocol #0 : iGEM General Protocol ==
 
#Obtaining Biobricks
 
#Obtaining Biobricks
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To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL
 
To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL
  
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==Protocol #2 : PCR and electrophoresis==
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==Protocol #3 : PCR and electrophoresis==
  
 
===PCR===
 
===PCR===
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| 95°C
 
| 95°C
 
| 10 sec
 
| 10 sec
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|rowspan="3"|X27
 
|-
 
|-
 
| Annealing
 
| Annealing
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===Electrophoresis===
 
===Electrophoresis===
  
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==Protocol #3 : Plasmid DNA purification==
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Preparation of 0.8% agarose gel
 +
 
 +
*Put 0.8g of agarose powder into 100mL of TAE 1X.
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*Melt in microwave and sitr occasionnally.
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*Let cool until you can take it into hands.
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*Flow the support with the gel.
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*Wait until it's solid.
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 +
Sample preparation and migration
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*Put the gel in the migration tank filled with 0.5X TAE.
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*Mix the samples with 1X marker***.
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*Migrate 30 min at 100V.
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 +
Revelation
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*Put the gel under UV.
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 +
==Protocol #4 : Plasmid DNA purification==
  
 
Protocol used for a Macherey-Nagel purification kit, for more details see [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf  here].
 
Protocol used for a Macherey-Nagel purification kit, for more details see [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf  here].
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Make sure to clear the lens with water and with a special paper  in order to no line the lens.
 
Make sure to clear the lens with water and with a special paper  in order to no line the lens.
  
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== Protocol #3 : Cloning protocol for IDT sequences ==
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== Protocol #5 : Cloning protocol for IDT sequences ==
 
===Resuspension===
 
===Resuspension===
 
Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer).
 
Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer).
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To make negative control, follow the same procedure but without add plasmids and spread 300 µL
 
To make negative control, follow the same procedure but without add plasmids and spread 300 µL
  
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== Protocol #4 : Digestion and ligation for verification and BioBrick Assembly ==
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== Protocol #6 : Digestion and ligation for verification and BioBrick Assembly ==
  
 
===Digestion (verification) protocol===
 
===Digestion (verification) protocol===
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Incubate at RT for 1 hour.
 
Incubate at RT for 1 hour.
  
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== #Protocol 5 : Generalised transduction using phage P1 ==
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== Protocol #7 : PCR clean-up and gel extraction ==
 +
 
 +
{| class="wikitable"
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|
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| PCR clean-up
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| Gel extraction
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|-
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|PCR clean-up,  DNA clean-up,<br/>or single stranded DNA clean-up: Adjust binding condition<br/><br/>Gel extraction: Excise DNA fragment / solubilize gel slice
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|200 μL NTI/ 100 μL PCR
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|200 μL NTI/ 100 mg gel<br/><br/>(melt at 50°C for 5–10 min)
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|-
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| Bind DNA
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| colspan="2"|Spin at 11,000g during 30s in a clean-up column
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|-
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| Wash silica membrane
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| colspan="2"|Add 700 μL NT3 and spin at 11,000g during 30s <u>Recommended</u>: 2nd wash again with 700μL NT3 spin 11,000g 30s
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|-
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| Dry silica membrane
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| colspan="2"|Spin at 11,000g during 1 min
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|-
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| Elute DNA
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| colspan="2"|Add 15–30μL NE, let at RT 1min and spin 11,000g during 1min
 +
|}
 +
 
 +
== Protocol #8 : Generalised transduction using phage P1 ==
  
  
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Transplant the good clones on LB-AB appropriate petri dishes
 
Transplant the good clones on LB-AB appropriate petri dishes
  
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== #Protocol 6 : Cadaverin HPLC analysis ==  
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== Protocol #9 : Cadaverin HPLC analysis ==  
  
 
===Reaction of lysine and cadaverine produced by biotransformation with whole cells===
 
===Reaction of lysine and cadaverine produced by biotransformation with whole cells===
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After derivatization with diethyl ethoxymethylmalonate, analyses were performed on a high performance liquid chromatograph (HPLC Agilent technologies, 1260 Infinity) consisting of a binary pump, an inline degasser, an autosampler and a column thermostat. Chromatographic separation was carried out by reverse-phasechromatography on a C18 column maintained at 35°C. Mobile phase A was composed of 100% acetonitrile, and B was made up of 25mM aqueous sodium acetate buffer (pH = 4.8). The flow rate of 1mL/min was used, with the following gradient program : 0-2min, (20-25% A; 2-32min, 25-60%A; 32-40min, 60-20%A. Detection was carried out at 284nm.
 
After derivatization with diethyl ethoxymethylmalonate, analyses were performed on a high performance liquid chromatograph (HPLC Agilent technologies, 1260 Infinity) consisting of a binary pump, an inline degasser, an autosampler and a column thermostat. Chromatographic separation was carried out by reverse-phasechromatography on a C18 column maintained at 35°C. Mobile phase A was composed of 100% acetonitrile, and B was made up of 25mM aqueous sodium acetate buffer (pH = 4.8). The flow rate of 1mL/min was used, with the following gradient program : 0-2min, (20-25% A; 2-32min, 25-60%A; 32-40min, 60-20%A. Detection was carried out at 284nm.
  
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== #Protocol 7 : SLIC (sequence- and ligation-independent cloning) ==  
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== Protocol #10 : SLIC (sequence- and ligation-independent cloning) ==  
  
 
===Procedures ===
 
===Procedures ===
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4. Add 0.2µL of T4 DNA polymerase (3U/µL, NEB) to the mixture and incubate 2min at room temperature
 
4. Add 0.2µL of T4 DNA polymerase (3U/µL, NEB) to the mixture and incubate 2min at room temperature
  
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== #Protocol 8 : Swimming test ==  
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== Protocol #11 : Swimming test ==  
  
 
Soft geloses have been made by adding 0.3% agar in LB medium. Once dried, each bacteria strain has been spotted using a tooth pic on the petri dishes and incubated for 3h in a 37°C room.
 
Soft geloses have been made by adding 0.3% agar in LB medium. Once dried, each bacteria strain has been spotted using a tooth pic on the petri dishes and incubated for 3h in a 37°C room.
  
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==SLIC Oligo table==
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== Protocol #12 : SDS page and coomassie blue ==  
  
 +
===Culture===
 +
 +
From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=1. Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min. After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer. We heated the mix at 95°C during 15min
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 +
===Staining with Coomassie Blue R250 ===
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 +
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* Stain the gel with 0.1% (or less) Coomassie Blue R250 in 10% acetic acid, 50% methanol, and 40% H2O for the minimum time (typically less than one hour) necessary to visualize the bands of interest.
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* The gel should be exposed to 10% acetic acid, 50% methanol for a total (stain plus destain) period of at least 3 hours (with shaking and at least three solvent changes) to ensure adequate removal of SDS.
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* Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear.
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 +
===Staining with Colloidal Coomassie Blue Staining Kit (Invitrogen LC6025) ===
 +
 +
• After electrophoresis, fix gel in 40% methanol/ 50%water/ 10% acetic acid for approximately ½ hr.  • Expose the gel in staining solution overnight and destain the gel by changing water frequently.
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 +
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{| class="wikitable"
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| Staining solution
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| 18 x 16cm per gel
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|-
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| Total volumes (ml)
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| 200
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|-
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| water
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| 110
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|-
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| MeOH
 +
| 10
 +
|-
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| Stainer A
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| 40
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|-
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| Stainer B
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| 10
 +
|}
 +
 +
Note: To get the highest sensitivity mix water, MeOH and Stainer A together and expose the gel in this solution for 10 min, then add the appropriate volume of Stainer B.
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 +
==SLIC Oligo table==
  
 
{| class="wikitable"
 
{| class="wikitable"
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|-
 
|-
 
| desB slic forward  
 
| desB slic forward  
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| "a rentrer"
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| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGGTATTGGTCTTGGG
 
|-
 
|-
 
| desB slic reverse  
 
| desB slic reverse  
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|-
 
|-
 
| desC slic forward  
 
| desC slic forward  
−
| "a rentrer"
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| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCTCGCCTTTCCACG
 
|-
 
|-
 
| desC slic reverse  
 
| desC slic reverse  
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|-
 
|-
 
| desD slic forward  
 
| desD slic forward  
−
| "a rentrer"
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| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGAGTTTAGCTGATGCA
 
|-
 
|-
 
| des D slic reverse   
 
| des D slic reverse   
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|-
 
|-
 
| CsgA E. Coli slic forward
 
| CsgA E. Coli slic forward
−
| "a rentrer"
+
| tttGAATTCGCGGCCGCTTCTAGatgaaacttttaaaagtagcagcaattg
 
|-
 
|-
 
| CsgA E. Coli slic reverse  
 
| CsgA E. Coli slic reverse  
−
| "A rentrer"
+
| aaaCTGCAGCGGCCGCTACTAGTAttattagtactgatgagcggtcgc
 
|}
 
|}
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−
 
  
 
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Latest revision as of 22:08, 19 October 2016