Difference between revisions of "Team:SJTU-BioX-Shanghai/Protocol"

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                     </div>
 
                     </div>
 
                     <div class="col-12 col-lg-9">
 
                     <div class="col-12 col-lg-9">
−
                         <div class="my-title h5-my-responsive" id="section1">Title One</div>
+
                         <div class="my-title h5-my-responsive" id="section1">PCR</div>
−
                         <p>Something something something something something something something something something</p>
+
                         <p class="my-title2">Materials:</p>
−
                         <p>Something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something</p>
+
                         <p>Buffer</br>Mg<sup>2+</sup></br>dNTP</br>Template</br>F/R-primer</br>DNA enzyme</p>
−
                         <div class="my-title h5-my-responsive" id="section2">Title Two</div>
+
                        <p class="my-title2">Methods:</p>
−
                         <p>Something something something something something something something something something</p>
+
                        <p>1.Combination of all reagents</p>
−
                         <p>Something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something</p>
+
                        <div class="figure-intro">
−
                         <div class="my-title h5-my-responsive" id="section3">Title Three</div>
+
                            <table class="table table-res table-res2 table-hover" align="center">
−
                         <p>Something something something something something something something something something</p>
+
 
−
                         <p>Something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something</p>
+
                                <thead>
−
                         <div class="my-title h5-my-responsive" id="section4">Title Four</div>
+
                                    <tr>
−
                         <p>Something something something something something something something something something</p>
+
                                        <td>Name of reagent</td>
−
                         <p>Something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something</p>
+
                                        <td>volume (μl)</td>
−
                         <div class="my-title h5-my-responsive" id="section5">Title Five</div>
+
                                    </tr>
−
                         <p>Something something something something something something something something something</p>
+
                                </thead>
−
                         <p>Something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something something</p>
+
                                <tbody>
−
                    </div>
+
                                    <tr>
 +
                                        <td>Buffer</td>
 +
                                        <td>2</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Mg<sup>2+</sup></td>
 +
                                        <td>2</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Template</td>
 +
                                        <td>20 ng for plasmid or 200 ng for genome</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>F/R-primer</td>
 +
                                        <td>0.6 for each</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Enzyme</td>
 +
                                        <td>0.4</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Sterile Water</td>
 +
                                        <td>make reaction up to 20μ in total</td>
 +
                                    </tr>
 +
                                </tbody>
 +
                            </table>
 +
                        </div>
 +
                        <p>2.Set the PCR machine to run the following steps:</p>
 +
                        <div class="figure-intro">
 +
                            <table class="table table-res table-res2 table-hover" align="center">
 +
 
 +
                                <thead>
 +
                                    <tr>
 +
                                        <td>Stage</td>
 +
                                        <td>Temperature (℃)</td>
 +
                                        <td>Time</td>
 +
                                    </tr>
 +
                                </thead>
 +
                                <tbody>
 +
                                    <tr>
 +
                                        <td>initial denaturation</td>
 +
                                        <td>94</td>
 +
                                        <td>2 min</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td> </td>
 +
                                        <td>94 (denaturation)</td>
 +
                                        <td>15 seconds</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>30 cycles</td>
 +
                                        <td>40-55 (annealing)</td>
 +
                                        <td>30 seconds</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td> </td>
 +
                                        <td>68 (extension)</td>
 +
                                        <td>30 seconds per bp</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Final extension</td>
 +
                                        <td>68</td>
 +
                                        <td>7 min</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Incubate</td>
 +
                                        <td>12</td>
 +
                                        <td>Infinite</td>
 +
                                    </tr>
 +
                                </tbody>
 +
                            </table>
 +
                        </div>
 +
 
 +
                         <div class="my-title h5-my-responsive" id="section2">Restriction Digestion</div>
 +
                         <p class="my-title2">Materials:</p>
 +
                         <p>Restriction Enzyme: Fisher FastDigest Restriction Enzyme</br>Green Buffer</br>DNA samplesr</br>Sterile Water</p>
 +
                        <p class="my-title2">Methods:</p>
 +
                        <p>Note: the components listed below are for double digestion!</p>
 +
                        <div class="figure-intro">
 +
                            <table class="table table-res table-res2 table-hover" align="center">
 +
                                <thead>
 +
                                    <tr>
 +
                                        <td>Component</td>
 +
                                        <td>volume (μl)</td>
 +
                                    </tr>
 +
                                </thead>
 +
                                <tbody>
 +
                                    <tr>
 +
                                        <td>Green Buffer</td>
 +
                                        <td>2</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>DNA sample</td>
 +
                                        <td>600 ng for plasmid or up to 200 ng for PCR production</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Template</td>
 +
                                        <td>20 ng for plasmid or 200 ng for genome</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Restriction Enzyme</td>
 +
                                        <td>1 for each</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Sterile Water</td>
 +
                                        <td>make reaction up to 20μ in total</td>
 +
                                    </tr>
 +
                                </tbody>
 +
                            </table>
 +
                        </div>
 +
                        <p>Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later</p>
 +
                         <div class="my-title h5-my-responsive" id="section3">Ligation</div>
 +
                         <p class="my-title2">Materials:</p>
 +
                         <p>T4 DNA Ligase</br>T4 DNA Ligase Buffer</br>Vector Plasmid</br>Insert DNA</br>Sterile Water</p>
 +
                        <p class="my-title2">Methods:</p>
 +
                        <div class="figure-intro">
 +
                            <table class="table table-res table-res2 table-hover" align="center">
 +
                                <thead>
 +
                                    <tr>
 +
                                        <td>Component</td>
 +
                                        <td>volume or mass</td>
 +
                                    </tr>
 +
                                </thead>
 +
                                <tbody>
 +
                                    <tr>
 +
                                        <td>T4 DNA Ligase Buffer</td>
 +
                                        <td>2 μ</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Vector Plasmid</td>
 +
                                        <td>50 ng</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Insert DNA</td>
 +
                                        <td>require molar ratio of 3:1 to 7:1 (insert:vector)</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>T4 DNA Ligase</td>
 +
                                        <td>0.2 μ</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <td>Sterile Water</td>
 +
                                        <td>make reaction up to 20μ in total</td>
 +
                                    </tr>
 +
                                </tbody>
 +
                            </table>
 +
                        </div>
 +
                        <p>Make the reaction and incubate at room temperature for 4-6 hours</p>
 +
                        <p>Note: for how to calculate volumes of vector and insert DNA, we highly recommend <a target="_blank" href="http://nebiocalculator.neb.com/#!/ligation">NEBbioCalculator</a></p>
 +
                        <p>Sterile Water</p>
 +
                         <div class="my-title h5-my-responsive" id="section4">DNA Gel Electrophoresis</div>
 +
                         <p class="my-title2">Materials:</p>
 +
                         <p>Agarose</br>TBE Buffer</br>Gel board</br>EB</br>DNA×10 or×6 Loading Buffer</br>Electrophoresis tank</br>sample</br>marker 2000 or 15000</p>
 +
 
 +
                        <p class="my-title2">Methods:</p>
 +
                        <p>1. Make a 1.0%-1.5% Agarose gel according to the volume of your gel board (eg. add 0.45g Agarose in 30ml TBE if choose a concentration of 1.5%)</p>
 +
                        <p>2. Heat the container with the mixture until it is complete dissolved. When the solution starts boiling, stop heating, shake the container until materials mix well and heat again until the mixture become clear</p>
 +
                        <p>3. Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead</p>
 +
                        <p>4. Pour the solution into a gel board and insert the comb</p>
 +
                        <p>5. Set the gel at room temperature and wait until it completely solidifies</p>
 +
                        <p>6. Remove the comb and transfer the gel to the electrophoresis tank</p>
 +
                        <p>7. Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)</p>
 +
                        <p>8. Run the gel for 20 min at 100V, 100mA</p>
 +
                         <div class="my-title h5-my-responsive" id="section5">Transformation</div>
 +
                         <p class="my-title2">Materials:</p>
 +
                         <p>LB broth</br>Ice</br>Selection plates with corresponding antibiotics.</p>
 +
 
 +
                        <div class="my-title h5-my-responsive" id="section6">Purification and Gel Extraction &amp; Plasmid Extraction</div>
 +
                        <div class="my-title h5-my-responsive" id="section7">Cotransformation</div>
 +
                        <div class="my-title h5-my-responsive" id="section8">Characterization</div>
  
 
                 </div>
 
                 </div>

Revision as of 03:22, 30 October 2017

Protocol
PCR

Materials:

Buffer
Mg2+
dNTP
Template
F/R-primer
DNA enzyme

Methods:

1.Combination of all reagents

Name of reagent volume (μl)
Buffer 2
Mg2+ 2
Template 20 ng for plasmid or 200 ng for genome
F/R-primer 0.6 for each
Enzyme 0.4
Sterile Water make reaction up to 20μ in total

2.Set the PCR machine to run the following steps:

Stage Temperature (℃) Time
initial denaturation 94 2 min
94 (denaturation) 15 seconds
30 cycles 40-55 (annealing) 30 seconds
68 (extension) 30 seconds per bp
Final extension 68 7 min
Incubate 12 Infinite
Restriction Digestion

Materials:

Restriction Enzyme: Fisher FastDigest Restriction Enzyme
Green Buffer
DNA samplesr
Sterile Water

Methods:

Note: the components listed below are for double digestion!

Component volume (μl)
Green Buffer 2
DNA sample 600 ng for plasmid or up to 200 ng for PCR production
Template 20 ng for plasmid or 200 ng for genome
Restriction Enzyme 1 for each
Sterile Water make reaction up to 20μ in total

Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later

Ligation

Materials:

T4 DNA Ligase
T4 DNA Ligase Buffer
Vector Plasmid
Insert DNA
Sterile Water

Methods:

Component volume or mass
T4 DNA Ligase Buffer 2 μ
Vector Plasmid 50 ng
Insert DNA require molar ratio of 3:1 to 7:1 (insert:vector)
T4 DNA Ligase 0.2 μ
Sterile Water make reaction up to 20μ in total

Make the reaction and incubate at room temperature for 4-6 hours

Note: for how to calculate volumes of vector and insert DNA, we highly recommend NEBbioCalculator

Sterile Water

DNA Gel Electrophoresis

Materials:

Agarose
TBE Buffer
Gel board
EB
DNA×10 or×6 Loading Buffer
Electrophoresis tank
sample
marker 2000 or 15000

Methods:

1. Make a 1.0%-1.5% Agarose gel according to the volume of your gel board (eg. add 0.45g Agarose in 30ml TBE if choose a concentration of 1.5%)

2. Heat the container with the mixture until it is complete dissolved. When the solution starts boiling, stop heating, shake the container until materials mix well and heat again until the mixture become clear

3. Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead

4. Pour the solution into a gel board and insert the comb

5. Set the gel at room temperature and wait until it completely solidifies

6. Remove the comb and transfer the gel to the electrophoresis tank

7. Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)

8. Run the gel for 20 min at 100V, 100mA

Transformation

Materials:

LB broth
Ice
Selection plates with corresponding antibiotics.

Purification and Gel Extraction & Plasmid Extraction
Cotransformation
Characterization
Wet Lab
Notebook
Wet Lab
Basic Parts