Team:DTU-Denmark/Methods/Protocols

From 2014.igem.org

(Difference between revisions)
Line 117: Line 117:
</div>
</div>
-
<h2 id="restriction-analysis-protocol" class="protocol-header">Restriction analysis</h2>
+
<h2 id="restriction-protocol" class="protocol-header">Restriction digestion</h2>
<div class="protocol restriction-analysis-protocol">
<div class="protocol restriction-analysis-protocol">
<ol>
<ol>
-
<li>Make a mastermix containing.</li>
+
    <li>Each reaction should contain:
-
  <ul>
+
        <ul>
-
             <li>1X NEBuffer (according to the desired restriction enzyme)</li>
+
             <li>1X NEBuffer (according to the desired restriction enzyme(s)) with BSA.</li>
-
            <li>1X BSA (note if NEBuffer is called X.1 BSA is already added in the buffer)</li>
+
             <li>1 unit/μl restriction enzyme.</li>
-
             <li>1 unit/μl restriction enzyme</li>
+
             <li>The DNA that is to be cut, final concentration of 10 ng/&mu;L</li>
-
              
+
            <li>Milli-Q water to a volume of 20 &mu;L</li>
-
                      </ul>
+
        </ul>
-
<li>Divide the mastermix into tubes and add DNA to a final concentration of 10 ng/μl</li>
+
    <li>Incubate the reactions at the restriction enzymes’ optimal temperature. If the restriction reaction is analytical, 30 minutes is enough. If the DNA is to be purified, a longer duration is recommended, e.g. 3 hours.</li>
-
<li>Incubate the reaction mix at the restriction enzymes optimal temperature for 30-60 minutes</li>
+
    <li>Add loading buffer to samples and run on 1% agarose gel with ethidium bromide (0.2 &mu;g/ml).</li>
-
<li>Add loading buffer to samples and run on 1% agarose gel with ethidium bromide 0.2 μg/ml</li>
+
    <li>Analyze your gel, and cut bands out if desired.</li>
-
<li>Analyze your gel</li>
+
</ol>
</ol>
-
</div>
 
-
 
-
<h2 id="standard-assembly-protocol" class="protocol-header">Standard assemby protocol</h2>
 
-
<div class="protocol standard-assembly-protocol">
 
-
http://parts.igem.org/Assembly:Standard_assembly<br>
 
-
Cut insert (“left fragment”) with EcoRI and SpeI.<br>
 
-
Cut backbone (and “right fragment”) with EcoRI and XbaI.<br>
 
-
 
-
 
-
----<br>
 
-
Enzyme restriction:<br>
 
-
 
-
Enzymes: 2x 0.5 µL<br>
 
-
Buffer: 2 µL (if it is 10x)<br>
 
-
DNA: ~1000-2000 ng (scale so the resulting fragments are equimolar)<br>
 
-
 
-
Total restriction mix: 20 µL (add MQ to volume)<br>
 
-
----
 
-
 
-
 
-
Purify the two fragments on a gel. Measure DNA concentration on the NanoDrop.<br>
 
-
 
-
Mix and ligate with T4 ligase.<br>
 
-
 
-
 
-
---<br>
 
-
Ligation protocol:<br>
 
-
 
-
T4 ligation buffer: 2 µL<br>
 
-
Ligase (add last): 1 µL<br>
 
-
DNA and MQ to 20 µL total volume<br>
 
-
Use http://django.gibthon.org/tools/ligcalc/ to calculate mixing proportions.<br>
 
-
 
-
Incubate at RT for ~2 h.<br>
 
-
---<br>
 
-
 
-
Transformation <br>
 
-
 
</div>
</div>
<h2 id="competent-cells-protocol" class="protocol-header">Preparation of competent cells</h2>
<h2 id="competent-cells-protocol" class="protocol-header">Preparation of competent cells</h2>
<div class="protocol competent-cells-protocol">
<div class="protocol competent-cells-protocol">
-
bla bla bla</br>
+
<ul>
-
la la la
+
    <li>2 mL LB is inoculated with cells from a single colony and incubated at 37 &deg;C O/N at 350 rpm</li>
 +
    <li>200 mL of preheated (37 &deg;C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 &deg;C and 350 rpm until OD 600 reaches 0.3 - 0.6</li>
 +
    <li>When OD600 reaches 0.3 - 0.6 the shake flask is chilled in an ice-bath until the culture is completely cooled. All steps from here should be kept as cold as possible.</li>
 +
    <li>The 200 mL culture is transferred to 50 mL tubes and spun at 5500 G for 10 minutes. The supernatants are discarded and each pellet is resuspended in 25 mL ice cold 0.1 Molar CaCl<sub>2</sub>. The tubes are poured together to 2x50 mL.</li>
 +
    <li>The cells are spun at 5500 G for 10 minutes and the supernatant is discarded. The cells are resuspended in 1 mL 0.1 Molar CaCl<sub>2</sub> and transferred to microcentrifuge tubes.</li>
 +
    <li>The cells are spun at 15000 G for 2 minutes and the supernatant is discarded. The cells are resuspended in 1 ml 0.1 Molar CaCl2 with 15% glycerol.</li>
 +
    <li>The cells are aliquoted into portions of 200 &mu;L in pre-chilled Eppendorf tubes and immediately stored at -80 &deg;C.</li>
 +
</ul>
</div>
</div>
<h2 id="chemical-transformation-protocol" class="protocol-header">Transformation with Chemically competent E. coli</h2>
<h2 id="chemical-transformation-protocol" class="protocol-header">Transformation with Chemically competent E. coli</h2>
<div class="protocol chemical-transformation-protocol">
<div class="protocol chemical-transformation-protocol">
-
<p>Transformation with Chemically competent E. coli<p><br>
+
Transformation with chemically competent <i>E. coli</i>
-
IMPORTANT.! Keep everything on ice unless otherwise stated in the protocol.<br>
+
IMPORTANT: Keep everything on ice unless otherwise stated in the protocol.
-
<ol>
+
<ul>
-
<li>Competent cells are defrosted in ice bath.</li>
+
    <li>Competent cells are defrosted in an ice bath</li>
-
 
+
    <li>50 &mu;L cells is transferred to a cooled eppendorf tube</li>
-
<li>40 μl cells is transferred to a cooled eppendorf tube.</li>
+
    <li>1-5 μl DNA is transferred to the competent cells, mixed by gently swirling the tube</li>
-
 
+
    <li>DNA and cell mix is kept on ice for 10 minutes</li>
-
<li>2 μl ice cold DNA is transferred to the competent cells, DNA and cells is mixed by inverting the tube 2-3 times.</li>
+
    <li>Cells are heat shocked at 42 &deg;C for 30 seconds in water bath</li>
-
 
+
    <li>Cells are kept on ice for 3 minutes.</li>
-
<li>DNA and cell mix is kept on ice for 10 minutes.</li>
+
    <li>500 &mu;L LB without antibiotics is added and incubated at 37 &deg;C for 30-120 minutes depending on antibiotic marker</li>
-
 
+
    <li>Plate cells on LB plate with the relevant antibiotic and incubate overnight at 37 &deg;C</li>
-
<li>Cells are heat shocked at 42 °C for 30 seconds in water bath for optimal heat transfer.</li>
+
</ul>
-
 
+
-
<li>Cells are kept on ice for 3 minutes.</li>
+
-
 
+
-
<li>Add 500 μl LB with no antibiotics and incubate cells at 37 °C for 30-60 minutes depending on antibiotic marker</li>
+
-
 
+
-
<li>Plate cells on LB plate with required antibiotic and incubate overnight at 37 °C.</li>
+
-
</ol>
+
</div>
</div>

Revision as of 13:15, 4 October 2014

Technical University of Denmark

Protocols

Glycerol stock-preservation

  1. Add 1 ml sterile 50% glycerol to a cryotube.
  2. Add 1 ml overnight culture to a cryotube from step 1
  3. Store at -80 °C

PCR


PCR mix calculator
Number of Reactions:
MQ water μL
HF buffer μL
dNTP μL
Polymerase μL
Template μL
Primer x2 μL
  • Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.
  • The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.
  • Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.
  • Mark the PCR tubes on lid and side and place in thermocycler.
  • The annealing temperature should match the primer melting temperatures minus 5 degrees. This can be hard since Tm predictors tend to disagree.
  • Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).

Gel electrophoresis

Preparation of Cam stock solution

Recipe on M9 minimal media

  1. Make M9 salts
  2. To make M9 Salts aliquot 800ml H2O and add
    • 64g Na2HPO4-7H2O
    • 15g KH2PO4
    • 2.5g NaCl
    • 5.0g NH4Cl
    • Stir until dissolved
    • Adjust to 1000ml with distilled H2O
    • Sterilize by autoclaving
  3. Measure ~700ml of distilled H2O (sterile)
  4. Add 200ml of M9 salts
  5. Add 2ml of 1M MgSO4 (sterile)
  6. Add 20 ml of 20% glucose (or other carbon source)
  7. Add 100ul of 1M CaCl2 (sterile)
  8. Adjust to 1000ml with distilled H2O

Restriction digestion

  1. Each reaction should contain:
    • 1X NEBuffer (according to the desired restriction enzyme(s)) with BSA.
    • 1 unit/μl restriction enzyme.
    • The DNA that is to be cut, final concentration of 10 ng/μL
    • Milli-Q water to a volume of 20 μL
  2. Incubate the reactions at the restriction enzymes’ optimal temperature. If the restriction reaction is analytical, 30 minutes is enough. If the DNA is to be purified, a longer duration is recommended, e.g. 3 hours.
  3. Add loading buffer to samples and run on 1% agarose gel with ethidium bromide (0.2 μg/ml).
  4. Analyze your gel, and cut bands out if desired.

Preparation of competent cells

  • 2 mL LB is inoculated with cells from a single colony and incubated at 37 °C O/N at 350 rpm
  • 200 mL of preheated (37 °C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 °C and 350 rpm until OD 600 reaches 0.3 - 0.6
  • When OD600 reaches 0.3 - 0.6 the shake flask is chilled in an ice-bath until the culture is completely cooled. All steps from here should be kept as cold as possible.
  • The 200 mL culture is transferred to 50 mL tubes and spun at 5500 G for 10 minutes. The supernatants are discarded and each pellet is resuspended in 25 mL ice cold 0.1 Molar CaCl2. The tubes are poured together to 2x50 mL.
  • The cells are spun at 5500 G for 10 minutes and the supernatant is discarded. The cells are resuspended in 1 mL 0.1 Molar CaCl2 and transferred to microcentrifuge tubes.
  • The cells are spun at 15000 G for 2 minutes and the supernatant is discarded. The cells are resuspended in 1 ml 0.1 Molar CaCl2 with 15% glycerol.
  • The cells are aliquoted into portions of 200 μL in pre-chilled Eppendorf tubes and immediately stored at -80 °C.

Transformation with Chemically competent E. coli

Transformation with chemically competent E. coli IMPORTANT: Keep everything on ice unless otherwise stated in the protocol.
  • Competent cells are defrosted in an ice bath
  • 50 μL cells is transferred to a cooled eppendorf tube
  • 1-5 μl DNA is transferred to the competent cells, mixed by gently swirling the tube
  • DNA and cell mix is kept on ice for 10 minutes
  • Cells are heat shocked at 42 °C for 30 seconds in water bath
  • Cells are kept on ice for 3 minutes.
  • 500 μL LB without antibiotics is added and incubated at 37 °C for 30-120 minutes depending on antibiotic marker
  • Plate cells on LB plate with the relevant antibiotic and incubate overnight at 37 °C

Transformation with electro-competent E. coli

Transformation with electro-competent E. coli


IMPORTANT.! Keep everything on ice unless otherwise stated in protocol.


Preparation: Put electro cuvettes, LB media with no antibiotics and DNA on ice in order to cool it down.

  1. Defrost electro-competent cells on ice
  2. Transfer 1 μl DNA to the competent cells
  3. Transfer DNA and cell mix to electroporation cuvette
  4. When ready the cuvette is quickly dried with paper towel and inserted to the electroporater and a shock is given. Immediately after the shock is given transfer 1 ml of ice cold LB to the cuvette and transfer the cells and media from the cuvette to an eppendorf tube and put the tube on ice.
  5. Leave the cells on ice for 10 minutes.
  6. Incubate the cells at 37 °C for 30-60 minutes depending on antibiotic marker
  7. Plate 100 μl on a half plate of LB with required antibiotic
  8. Spin the cells briefly at maximum RPM and decant the supernatant gently, approximately 100 μl will remain in the tube, resuspend the pellet in the rest of the supernatant and plate it on the other half of the plate used in step 7.
  9. Incubate plates at 37 °C overnight
Technical University of Denmark
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark
+45 45 25 25 25
dtuigem14@gmail.com .

Retrieved from "http://2014.igem.org/Team:DTU-Denmark/Methods/Protocols"