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

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{{Aix-Marseille}}
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= Protocols =
+
 
== Protocol #0 : iGEM General Protocol ==
 
== Protocol #0 : iGEM General Protocol ==
 
#Obtaining Biobricks
 
#Obtaining Biobricks
Line 51: Line 50:
 
== Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers ==
 
== Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers ==
  
−
For 200 mL of culture:
+
For 100 mL of culture:
  
 
===Preparation of 50 mL of Tbf1 Buffer===
 
===Preparation of 50 mL of Tbf1 Buffer===
Line 98: Line 97:
 
== Protocol #2 : Transformations ==
 
== Protocol #2 : Transformations ==
 
===Plasmids transformation ===
 
===Plasmids transformation ===
−
#Add 20 ng {of plasmid to 100 μL of competent cells thawed in ice
+
#Add 20 ng of plasmid to 100 μL of competent cells thawed in ice
 
#Incubate 30-45 min in ice
 
#Incubate 30-45 min in ice
 
#Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
 
#Thermal shock : put tubes in the Thermomixer at 42°C for 2 min
Line 119: Line 118:
 
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
  
−
== Protocol #3 : Cloning protocol for IDT sequences ==
+
==Protocol #3 : PCR and electrophoresis==
 +
 
 +
===PCR===
 +
 
 +
{| class="wikitable"
 +
|
 +
| 25µL reaction
 +
| 50µL reaction
 +
| 100µL reaction
 +
| Final<br/>concentration
 +
|-
 +
| EconoTaq PLUS<br/>2X Master Mix
 +
| 12.5µL
 +
| 25µL
 +
| 50µL
 +
| 1X
 +
|-
 +
|Forward Primer<br/>(100 pmol/μL)
 +
| 0.5µL
 +
| 0.5µL
 +
| 1µL
 +
| 1 pmol/μL (1 μM)
 +
|-
 +
|Reverse Primer<br/>(100 pmol/μL)
 +
| 0.5µL
 +
| 0.5µL
 +
| 1µL
 +
| 1 pmol/μL (1 μM)
 +
|-
 +
|DNA Template*
 +
| 0.5µL
 +
| 1µL
 +
| 1µL
 +
| ?
 +
|-
 +
|Water<br/>nuclease-free
 +
| [ ] μL qsp 25μL
 +
| [ ] μL qsp 50µL
 +
| [ ] μL qsp 100μL
 +
|
 +
|}
 +
 
 +
* *One colony in 50 μL of Water. 15-20 min at 80°C. 5 min at 96°C.
 +
 
 +
Cycling steps
 +
 
 +
{| class="wikitable"
 +
| Cycling step
 +
| Temperature
 +
| Time
 +
| Number of cycles
 +
|-
 +
| Initial denaturation
 +
| 98°C
 +
| 3 min
 +
| 1
 +
|-
 +
| Denaturation
 +
| 95°C
 +
| 10 sec
 +
|rowspan="3"|X27
 +
|-
 +
| Annealing
 +
| 50-65°C
 +
| 40 sec
 +
|-
 +
| Extension
 +
| 72°C
 +
| 1 min/kb
 +
|-
 +
| Final extension
 +
| 72°C
 +
| 1 min/kb
 +
| 1
 +
|-
 +
|}
 +
 
 +
===Electrophoresis===
 +
 
 +
Preparation of 0.8% agarose gel
 +
 
 +
*Put 0.8g of agarose powder into 100mL of TAE 1X.
 +
*Melt in microwave and sitr occasionnally.
 +
*Let cool until you can take it into hands.
 +
*Flow the support with the gel.
 +
*Wait until it's solid.
 +
 
 +
Sample preparation and migration
 +
 
 +
*Put the gel in the migration tank filled with 0.5X TAE.
 +
*Mix the samples with 1X marker***.
 +
*Migrate 30 min at 100V.
 +
 
 +
Revelation
 +
 
 +
*Put the gel under UV.
 +
 
 +
==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].
 +
For better results use gloves and if so do not use gloves.
 +
 
 +
{| class="wikitable"
 +
|'''Step'''
 +
|'''Actions'''
 +
|-
 +
|Cultivate and harvest cells
 +
|Centrifuge at 11,000g for 30 sec
 +
|-
 +
|Cell lysis
 +
|Add 250µL of buffer A1 <br/> Add 250µL of buffer A2 <br/> Incubate at room temperature up to 5 min <br/> 300µL buffer A3
 +
|-
 +
|Clarification of the lysate
 +
|Centrifuge at 11,000g for 5 min
 +
|-
 +
|Bind DNA
 +
|Place a purification column in a collection tube (2mL) and  carefully decant supernatant from the previous step, to a maximum of 750µL <br/> Then centrifuge at 11,000g for 1 min and discard the flow-through. <br/> Put the column back in the collection tube <br/> Repeat this step to load the rest of the supernatant if need be
 +
|-
 +
|Wash silica membrane
 +
|Add 600µl of buffer A4 <br/> Centrifuge at 11,000g for 1 min
 +
|-
 +
|Dry silica membrane
 +
|Centrifuge at 11,000g for 2 min
 +
|-
 +
|Elute DNA
 +
|Place the columns in an 1.5mL eppendorf and add 50 µL of buffer AE and incubate at room temperature for 1 min <br/> Centrifuge at 11,000g for 1 min
 +
 
 +
|}
 +
 
 +
To measure your DNA concentration, use a Biospec Nano apparel or Nanodrop apparel.
 +
Make the blank with  1µL of pure water and use 1µl of sample.
 +
Make sure to clear the lens with water and with a special paper  in order to no line the lens.
 +
 
 +
== 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).
Line 191: Line 323:
 
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
  
−
== Protocol #4 : Digestion and ligation for verification and BioBrick Assembly ==
+
== Protocol #6 : Digestion and ligation for verification and BioBrick Assembly ==
  
 
===Digestion (verification) protocol===
 
===Digestion (verification) protocol===
Line 207: Line 339:
 
|10X NEBuffer 2
 
|10X NEBuffer 2
 
|2 µL
 
|2 µL
−
|-
 
−
|100X BSA
 
−
|0.2 µL
 
 
|-
 
|-
 
|H2O  
 
|H2O  
Line 232: Line 361:
 
|10X NEBuffer 2
 
|10X NEBuffer 2
 
|5 µL
 
|5 µL
−
|-
 
−
|100X BSA
 
−
|0.5 µL
 
 
|-
 
|-
 
|H2O
 
|H2O
 
|To 50 µL
 
|To 50 µL
 
|}
 
|}
−
====Downstream part :====
+
====Downstream part, optional, for 3A strategy ====
 
{| class="wikitable"
 
{| class="wikitable"
 
|Downstream part plasmid
 
|Downstream part plasmid
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|10X NEBuffer 2
 
|10X NEBuffer 2
 
|5 µL
 
|5 µL
−
|-
 
−
|100X BSA
 
−
|0.5 µL
 
 
|-
 
|-
 
|H2O
 
|H2O
Line 309: Line 432:
  
 
Incubate at RT for 1 hour.
 
Incubate at RT for 1 hour.
 +
 +
== Protocol #7 : PCR clean-up and gel extraction ==
 +
 +
{| class="wikitable"
 +
|
 +
| PCR clean-up
 +
| Gel extraction
 +
|-
 +
|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
 +
|200 μL NTI/ 100 μL PCR
 +
|200 μL NTI/ 100 mg gel<br/><br/>(melt at 50°C for 5–10 min)
 +
|-
 +
| Bind DNA
 +
| colspan="2"|Spin at 11,000g during 30s in a clean-up column
 +
|-
 +
| Wash silica membrane
 +
| 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
 +
|-
 +
| Dry silica membrane
 +
| colspan="2"|Spin at 11,000g during 1 min
 +
|-
 +
| Elute DNA
 +
| colspan="2"|Add 15–30μL NE, let at RT 1min and spin 11,000g during 1min
 +
|}
 +
 +
== Protocol #8 : Generalised transduction using phage P1 ==
 +
 +
 +
===Step1 : P1 lysat on the given strain ===
 +
 +
From a overnight starter, (3mL in 2YT medium + appropriated antibiotiques) from the strand holding the transduct mutation, make a 1/100 dilution in 3mL 2YT medium and add 150µL of Cacl2 0.1M ( without antibiotique corresponding of the transduct allele).
 +
Incubate at 37°C (under agitation) until obtening a DO600=0.5-1 (more 0.5)
 +
Add 150µL of a P1 lysat wt
 +
Incubate at 37°C without agitation (water bath) during 20min
 +
Incubate at 37°C under agitation during 3 or 4h (even overnight)
 +
Divide the culture in 2 eppendorfs of 2mL (=2*1.5mL)
 +
Add 150µL chloroform then vortex
 +
Centrifuge 10min at 4°C and 6000rpm
 +
Transfer 1mL supernatant into 2 news eppendorf of 2mL
 +
Add 10µL chloroform (under a fumehood) in each eppendorf, vortex and keep at 4°C
 +
(option: to increase the titration level, you can do a second cycle)
 +
 +
===Step2 : Transduction P1===
 +
 +
The eve, prepare a culture of the recipient strain in 3mL 2YT medium (+eventual appropriated antibiotique)
 +
The D-day, start a culture from the recipient strain (1/50 dilution) in 3mL 2YT + 150µL of CaCl2 0.1M
 +
( prepare the LB-citrate 5mM requiered and LBagar- appropriated AB + citrate 2mM petri dishes)
 +
Incubate until reaching a DO600 = 1 or + (2.10^6 bact/mL)
 +
Prepare 3 tubes eppendorf of 2mL (1 control with 500µL cells, 1 with 10µL P1 + 500µL cells, 1 with 100µL P1 + 500µL cells)
 +
Infect during 20min at 37°C (heat wuthout agitation)
 +
Add 1mL steril LB-citrate 5mM and heat at 37°C during 50min under agitation (700-750rpm)
 +
Centrifuge 5min at 5000rpm (RT)
 +
Remove supernatant sterilely with a P1000 and resuspend in 1mL steril LB-citrate 5mM
 +
Spread on LBagar- appropried AB + 2mM citrate
 +
Incubate ON at 37°C
 +
 +
===Step3 : Clones isolation===
 +
 +
Isolate clones on petri dishes (approprieted AB + citrate 2mM)
 +
Incubate at 37°C
 +
 +
===Step 4 : PCR screen and transplantation===
 +
 +
Test clones by PCR
 +
Transplant the good clones on LB-AB appropriate petri dishes
 +
 +
== Protocol #9 : Cadaverin HPLC analysis ==
 +
 +
===Reaction of lysine and cadaverine produced by biotransformation with whole cells===
 +
 +
The assays were performed in a total volume of 500µL, containing 500mM sodium acetate buffer (ph = 6.0), 10mM L-lysine, 0.1 mM pyridoxal-5-phosphate, and 20µL whole cells, at 37°C in a water bath. The reaction was stopped after 24h by the addition of 10µL ethanol. The reaction mixtures were centrifuged and the 30µL of supernatant was applied to derivatization to determine the amounts of residual lysine and product, i.e cadaverine.
 +
 +
===Derivatization reaction===
 +
 +
Diamine deritatives were obtained by the reaction of 180µL of borate buffer 0.05M (pH9), 60µL of 100% methanol, 47µL diethyl ethoxymethylenemalonate without any pretreatment. The samples were heated at 70)C for 2h to allow complete degradation of excess DEEMM and derivatization.
 +
 +
===HPLC analysis===
 +
 +
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.
 +
 +
== Protocol #10 : SLIC (sequence- and ligation-independent cloning) ==
 +
 +
===Procedures ===
 +
 +
1. Digest vector with restriction enzyme during 3h and purify the linearized vector with a commercial PCR purification kit. Elute DNA with elution buffer or 10mM TrisCl, pH 8.0-8.5. Do nnot elute the DNA with water or TE. Measure the concentration of the vector. 
 +
 +
2. Amplify your gene of interest by PCR using primers with 15 mer (or inferior) homology extension to the linearized vector end. We usually use 15bp homology for single fragment cloning, and 20bp for multiple fragment cloning. Purify the linearized vector with a commercial PCR purification kit. Elute the DNA with water or TE. Measure the concentration of the insert(s).
 +
 +
3. Mix the linearized vector and insert at a molar ratio of 1.2 ina 1.5mL tube. An examble is shown as follows. (Vector to insert molar ratio of 1/1 for single fragment cloning, 1:2:2 for multiple fragments cloning. AN example of 3 fragments cloning is shown below with vector: insert 1: insert 2 molar ratio as shown is 1:2:2 as shown in Fig. 4A).
 +
 +
{| class="wikitable"
 +
|
 +
| Stock concentration
 +
| Volume added
 +
| Final concentration
 +
|-
 +
| Linearized vector
 +
| 100ng/µL
 +
| 1µL
 +
| 10ng/µL
 +
|-
 +
| Insert 1
 +
| 40ng/µL
 +
| 1µL
 +
| 4ng/µL
 +
|-
 +
| Insert 2
 +
| 40ng/µL
 +
| 1µL
 +
| 4ng/µL
 +
|-
 +
| 10X BSA
 +
|
 +
| 1µL
 +
| 1X
 +
|-
 +
| 10X NEB Buffer 2
 +
|
 +
| 1µL
 +
| 1X
 +
|-
 +
| H2O
 +
|
 +
| Up to 10µL
 +
|
 +
|}
 +
 +
4. Add 0.2µL of T4 DNA polymerase (3U/µL, NEB) to the mixture and incubate 2min at room temperature
 +
 +
== 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.
 +
 +
== 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
 +
 +
===Staining with Coomassie Blue R250 ===
 +
 +
 +
* 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.
 +
 +
* 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.
 +
 +
* 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.
 +
 +
===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.
 +
 +
 +
{| class="wikitable"
 +
| Staining solution
 +
| 18 x 16cm per gel
 +
|-
 +
| Total volumes (ml)
 +
| 200
 +
|-
 +
| water
 +
| 110
 +
|-
 +
| MeOH
 +
| 10
 +
|-
 +
| Stainer A
 +
| 40
 +
|-
 +
| Stainer B
 +
| 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.
 +
 +
==SLIC Oligo table==
 +
 +
{| class="wikitable"
 +
| DesA slic forward
 +
| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGCGCTCGCACTTGCTT
 +
|-
 +
| desA slic reverse
 +
| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGAGGCACGGTC
 +
|-
 +
| desB slic forward
 +
| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGGTATTGGTCTTGGG
 +
|-
 +
| desB slic reverse
 +
| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTACACTGCAAATTC
 +
|-
 +
| desC slic forward
 +
| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCTCGCCTTTCCACG
 +
|-
 +
| desC slic reverse
 +
| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGCTGAAACCGC
 +
|-
 +
| desD slic forward
 +
| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGAGTTTAGCTGATGCA
 +
|-
 +
| des D slic reverse 
 +
| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACGCCCGGCTAA
 +
|-
 +
| FliC E. coli slic forward 
 +
| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT
 +
|-
 +
| FliC E.coli slic reverse
 +
| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG
 +
|-
 +
| FliC desulfo slic forward
 +
| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCACTGGTTATCAAT
 +
|-
 +
| FliC desulfo slic reverse
 +
| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGCCGCCGAGAAG
 +
|-
 +
| CsgA E. Coli slic forward
 +
| tttGAATTCGCGGCCGCTTCTAGatgaaacttttaaaagtagcagcaattg
 +
|-
 +
| CsgA E. Coli slic reverse
 +
| aaaCTGCAGCGGCCGCTACTAGTAttattagtactgatgagcggtcgc
 +
|}
 +
 +
{{:Team:Aix-Marseille/Template-Footer}}

Latest revision as of 22:08, 19 October 2016