Difference between revisions of "Team:Tuebingen/Methods"

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<li>transfer into 50mL Falcon tubes</li>
 
<li>transfer into 50mL Falcon tubes</li>
 
<li>refrigerate centrifuge at 4&#176;C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol</li>
 
<li>refrigerate centrifuge at 4&#176;C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol</li>
−
<li>spin down cells at 2000g for 10Min at 4&#176;C</li>
+
<li>spin down cells at 2,000g for 10Min at 4&#176;C</li>
 
<li>resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2</li>
 
<li>resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2</li>
−
<li>spin down at 2000g for 10Min at 4&#176;C</li>
+
<li>spin down at 2,000g for 10Min at 4&#176;C</li>
 
<li>resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol</li>
 
<li>resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol</li>
 
<li>freeze 50-100&#956;l aliquots</li>
 
<li>freeze 50-100&#956;l aliquots</li>
Line 93: Line 93:
 
<li>Pick colonies for overnight cultures in 23ml LB medium with antibiotic
 
<li>Pick colonies for overnight cultures in 23ml LB medium with antibiotic
 
grow overnight</li>
 
grow overnight</li>
−
<li>Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and discard supernatant</li>
+
<li>Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1.5ml, spin again and discard supernatant</li>
 
<li>Resuspend the bacteria pellet in 600&#956;l H2O</li>
 
<li>Resuspend the bacteria pellet in 600&#956;l H2O</li>
 
<li>Add 100 &#956;l Cell Lysis Buffer, invert six times</li>
 
<li>Add 100 &#956;l Cell Lysis Buffer, invert six times</li>
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<div class="contentRow">
 
<div class="contentRow">
 
<div class="contentCell contentSmallCell">
 
<div class="contentCell contentSmallCell">
−
Transformation of Bacteria
+
Transformation of E. coli
 
</div>
 
</div>
 
<div class="contentCell">
 
<div class="contentCell">
Line 139: Line 139:
 
<li> spin down bacteria at 2g for 2min</li>
 
<li> spin down bacteria at 2g for 2min</li>
 
<li> discard supernatant by tipping the tube</li>
 
<li> discard supernatant by tipping the tube</li>
−
<li> resuspend bacterial pellet in leftover supernatant (50100&#956;l)</li>
+
<li> resuspend bacterial pellet in leftover supernatant (50-100&#956;l)</li>
 
<li> streak bacteria onto LBagar plates with antibiotics and incubate overnight </li>
 
<li> streak bacteria onto LBagar plates with antibiotics and incubate overnight </li>
 +
</div>
 +
</div>
 +
<div class="contentRow">
 +
<div class="contentCell contentSmallCell">
 +
Transformation of L. johnsonii
 +
</div>
 +
<div class="contentCell">
 +
<li>100ml <em>L. johnsonii (OD595=0.7)</em></li>
 +
<li>107ml Transformation buffer</li>
 +
<li>10&#956;l vector DNA
 +
<p>Method:</p>
 +
<li> Resuspend pelleted bacteria (4000xg/5min) in 100ml Transformation buffer </li>
 +
<li> Zentrifuge and resuspend the bacteria again in 5ml Transformation buffer</li>
 +
<li> Repaet step with resuspension in 2ml Transformation buffer</li>
 +
<li> Hold on ice for 1min</li>
 +
<li> Electroporate 200&#956;l bacteria in 0.2cm cuvette at 25&#956;F and 200ohms and time constant between 4.1 and 4.5ms</li>
 +
<li> Mix electroporated bacteria with 1ml MRS medium each </li>
 +
<li> Incubate each bacteria solution in additional 15ml MRS medium for 18h at 37&#176;C </li>
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="contentRow">
 
<div class="contentRow">
 
<h1>Materials</h1>
 
<h1>Materials</h1>
 +
</div>
 +
<div class="contentRow">
 +
<div class="contentCell contentSmallCell">
 +
50x TAE buffer
 +
</div>
 +
<div class="contentCell">
 +
<li>121g Tris base</li>
 +
<li>28.5ml Acetic acid</li>
 +
<li>9.3g EDTA</li>
 +
<li>to 500ml with H2O</li>
 +
</div>
 
</div>
 
</div>
 
<div class="contentRow">
 
<div class="contentRow">
Line 164: Line 193:
 
<li>15g Agar</li>
 
<li>15g Agar</li>
 
<li>to 1l with H2O</li>
 
<li>to 1l with H2O</li>
 +
</div>
 +
</div>
 +
<div class="contentRow">
 +
<div class="contentCell contentSmallCell">
 +
MRS medium
 +
</div>
 +
<div class="contentCell">
 +
<p>Medium prepared as described by manufacturer</p>
 
</div>
 
</div>
 
</div>
 
</div>
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<li>chloramphenicol 34µg/ml</li>
 
<li>chloramphenicol 34µg/ml</li>
 
<li>ampicillin 100µg/ml</li>
 
<li>ampicillin 100µg/ml</li>
 +
</div>
 +
</div>
 +
<div class="contentRow">
 +
<div class="contentCell contentSmallCell">
 +
Transformation buffer for L. johnsonii
 +
</div>
 +
<div class="contentCell">
 +
<li>342.3g Sucrose</li>
 +
<li>0.277g CaCl2</li>
 +
<li>to 1l H2O</li>
 +
<li></li>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</html>
 
</html>

Revision as of 16:49, 19 October 2016

Methods

Chemically Competent E. coli Cells
  • autoclave 250mL LB in Erlenmeyer beaker
  • plate cells from cell stock on agarose plate with appropriate antibiotics
  • inoculate one clone in 5mL of LB with antibiotics, grow at 37°C o/n
  • expand to 500mL and grow until OD600nm = 0.35
  • transfer into 50mL Falcon tubes
  • refrigerate centrifuge at 4°C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol
  • spin down cells at 2,000g for 10Min at 4°C
  • resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2
  • spin down at 2,000g for 10Min at 4°C
  • resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol
  • freeze 50-100μl aliquots
  • store at 20°C
    • DoubleDigest Restriction of gBlocks and BioBricks
  • 2μl 10x buffer (corresponding to enzymes)
  • 0,5μl restriction enzyme 1
  • 0,5μl restriction enzyme 2
  • 1μg DNA
  • fill up to 20μl with H2O

    • Method:

  • mix all components
  • incubate at 37°C for 2h
  • heatinactivate enzymes at 80°C for 10min or use for gel purification
    • Gelpurification

    This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega

  • Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube
  • Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060°C until gel slice is completely dissolved
  • Insert SV Minicolumn into Collection Tube
  • Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute
  • Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube
  • Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min
  • Discard flowthrough and reinsert Minicolumn into Collection tube
  • Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min
  • Empty the Collection Tube and centrifuge the column assembly for 1,5 min
  • Leave the tubes open for 10 min (to let any rest of ethanol evaporate)
  • Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube
  • Add 30μl of NucleaseFreeWater (65°C) to the Minicolumn. Incubate at room temperature for 10 min
  • Centrifuge at 16,000xg for 1 min.
  • Incubate sample at 65°C for 5min
  • Discard Minicolumn and store DNA at 4°C or 20°C
    • Ligation
  • 1μl 10x ligase buffer
  • 0,5μl ligase
  • 1μl ATP
  • 0,51μl linearised vector
  • 7μl insert

    • Method:

  • Mix all components
  • Incubate overnight at 4°C
  • Gelpurify using agarose gels
    • Smallscale plasmid preparation

    Preparation was done using the PureYield™ Plasmid Miniprep System by Promega

  • Pick colonies for overnight cultures in 23ml LB medium with antibiotic grow overnight
  • Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1.5ml, spin again and discard supernatant
  • Resuspend the bacteria pellet in 600μl H2O
  • Add 100 μl Cell Lysis Buffer, invert six times
  • Add 350 μl cold Neutralization Solution, mix thoroughly by inverting
  • Centrifuge 30s at maximum speed
  • Transfer supernatant to PureYield Minicolumn in collection tube
  • Centrifuge for 30 sec, discard the flow-through
  • Add 200 μl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.
  • Add 400 μl Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.
  • Place column in eppendorf tube, add 30 μl Elution Buffer to minicolumn matrix, incubate for 1 min
  • Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA
  • Measure the DNA concentration using a Nanodrop
    • Testrestriction
  • 0,25μl Enzyme 1
  • 0,25μl Enzyme 2
  • 1μl 10x buffer (corresponding to enzymes)
  • 200-300ng DNA
  • Fill up with water to 10μl.

    • Method:

  • Incubate at 37°C for 1h
  • Run on 1% agarose gel
    • Transformation of E. coli
  • 50μl chemically competent E. coli
  • 35μl overnight ligation mix OR 1ng purified plasmid DNA

    • Method:

  • Thaw bacteria pellet on ice
  • add ligation mixture or purified plasmid to bacteria
  • incubate for 20min on ice
  • heat shock bacteria for 45s at 42°C
  • incubate bacteria on ice for 2min
  • add 700μl LB medium without antibiotics
  • incubate for 1h at 37°C
  • spin down bacteria at 2g for 2min
  • discard supernatant by tipping the tube
  • resuspend bacterial pellet in leftover supernatant (50-100μl)
  • streak bacteria onto LBagar plates with antibiotics and incubate overnight
    • Transformation of L. johnsonii
  • 100ml L. johnsonii (OD595=0.7)
  • 107ml Transformation buffer
  • 10μl vector DNA

    Method:

  • Resuspend pelleted bacteria (4000xg/5min) in 100ml Transformation buffer
  • Zentrifuge and resuspend the bacteria again in 5ml Transformation buffer
  • Repaet step with resuspension in 2ml Transformation buffer
  • Hold on ice for 1min
  • Electroporate 200μl bacteria in 0.2cm cuvette at 25μF and 200ohms and time constant between 4.1 and 4.5ms
  • Mix electroporated bacteria with 1ml MRS medium each
  • Incubate each bacteria solution in additional 15ml MRS medium for 18h at 37°C

    • Materials

    50x TAE buffer
  • 121g Tris base
  • 28.5ml Acetic acid
  • 9.3g EDTA
  • to 500ml with H2O
    • LB
  • 25g Lennox Broth
  • to 1l with H2O
    • LB-Agar
  • 25g Lennox Broth
  • 15g Agar
  • to 1l with H2O
    • MRS medium

    Medium prepared as described by manufacturer

    antibiotics concentration
  • chloramphenicol 34µg/ml
  • ampicillin 100µg/ml
    • Transformation buffer for L. johnsonii
  • 342.3g Sucrose
  • 0.277g CaCl2
  • to 1l H2O