Team:Nanjing-China/protocol

From 2013.igem.org

(Difference between revisions)
Line 122: Line 122:
             <dt><a href="###">Agarose gel electrophoresis</a></dt>
             <dt><a href="###">Agarose gel electrophoresis</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.<br>
-
(1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.
+
(2) Melt the mixture in a microwave until the solution becomes clear.<br>
-
(2) Melt the mixture in a microwave until the solution becomes clear.
+
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.<br>
-
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.
+
(4) Pour the solution into the gel casting tray with appropriate comb.<br>
-
(4) Pour the solution into the gel casting tray with appropriate comb.
+
(5) Let the gel cool until it is solid.<br>
-
(5) Let the gel cool until it is solid.
+
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.<br>
-
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.
+
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.<br>
-
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.
+
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.<br>
-
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.
+
(9) Run the gel at 135V for about 20min.
(9) Run the gel at 135V for about 20min.
-
</pre>            
+
            
             </dd>
             </dd>
         </dl>
         </dl>
Line 139: Line 138:
             <dt><a href="###">Gel purification with Axygen Gel Extraction Kit</a></dt>
             <dt><a href="###">Gel purification with Axygen Gel Extraction Kit</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
 
-
(1) Excise the DNA fragment from the agarose gel with a clean shaver blade.
 
-
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.
 
-
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.
 
-
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.
 
-
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.
 
-
(6) Apply the solution from the last step to the column.
 
-
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.
 
-
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.
 
-
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.
 
-
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.
 
-
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.
 
-
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).
 
-
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).
 
-
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).
 
-
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.
 
-
</pre>           
+
(1) Excise the DNA fragment from the agarose gel with a clean shaver blade.<br>
 +
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.<br>
 +
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.<br>
 +
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.<br>
 +
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.<br>
 +
(6) Apply the solution from the last step to the column.<br>
 +
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.<br>
 +
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.<br>
 +
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.<br>
 +
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.<br>
 +
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br>
 +
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br>
 +
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).<br>
 +
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br>
 +
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br>
 +
            
             </dd>
             </dd>
         </dl>
         </dl>
Line 163: Line 161:
             <dt><a href="###">Ligation</a></dt>
             <dt><a href="###">Ligation</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Test the concentration of the DNA samples.<br>
-
(1) Test the concentration of the DNA samples.
+
(2) Pipet the following into a PCR tube:<br>
-
(2) Pipet the following into a PCR tube:
+
     Linearized vector DNA <br>
-
     Linearized vector DNA  
+
     Insert DNA<br>
-
     Insert DNA
+
     Solution I<br>
-
     Solution I
+
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.<br>
-
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.
+
(4) Pause at 4℃. <br>        
-
(4) Pause at 4℃.
+
-
 
+
-
</pre>          
+
             </dd>
             </dd>
         </dl>  
         </dl>  
Line 179: Line 174:
             <dt><a href="###">Tranformatiom</a></dt>
             <dt><a href="###">Tranformatiom</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Get the competent cell from -80℃ and wait for its fusion on ice.<br>
-
(1) Get the competent cell from -80℃ and wait for its fusion on ice.
+
(2) Add the entire ligation product or 1μL plasmid into the tube.<br>
-
(2) Add the entire ligation product or 1μL plasmid into the tube.
+
(3) Mix and incubate on ice for 30min.<br>
-
(3) Mix and incubate on ice for 30min.
+
(4) Heat pulse at 42℃ for 90sec.<br>
-
(4) Heat pulse at 42℃ for 90sec.
+
(5) Put back the tube on ice and incubate for 5min.<br>
-
(5) Put back the tube on ice and incubate for 5min.
+
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.<br>
-
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.
+
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.<br>
-
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.
+
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics. <br>
-
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics.
+
-
</pre>          
+
             </dd>
             </dd>
         </dl>  
         </dl>  
Line 195: Line 188:
             <dt><a href="###">Mini-prep with Axygen Mini-prep Kit</a></dt>
             <dt><a href="###">Mini-prep with Axygen Mini-prep Kit</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.<br>
-
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.
+
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.<br>
-
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.
+
(3) Repeat the last step.<br>
-
(3) Repeat the last step.
+
(4) Resuspend compact cells in 250μL buffer S1.<br>
-
(4) Resuspend compact cells in 250μL buffer S1.
+
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.<br>
-
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.
+
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.<br>
-
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.
+
(7) Centrifuge at 12000rpm for 10min.<br>
-
(7) Centrifuge at 12000rpm for 10min.
+
(8) Apply the supernatant from the last step to the column.<br>
-
(8) Apply the supernatant from the last step to the column.
+
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.<br>
-
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.
+
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br>
-
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.
+
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.<br>
-
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.
+
(12) Repeat the last step.<br>
-
(12) Repeat the last step.
+
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.<br>
-
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.
+
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).<br>
-
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).
+
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).<br>
-
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).
+
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).<br>
-
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).
+
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.<br>        
-
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.
+
-
</pre>          
+
             </dd>
             </dd>
         </dl>
         </dl>
Line 220: Line 211:
             <dt><a href="###">Preparation of competent cells</a></dt>
             <dt><a href="###">Preparation of competent cells</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.<br>
-
(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.
+
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.<br>
-
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.
+
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br>
-
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.
+
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.<br>
-
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.
+
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.<br>
-
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.
+
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.<br>
-
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.
+
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.<br>
-
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.
+
(8) Store at -80℃ after liquid nitrogen freezing.<br>
-
(8) Store at -80℃ after liquid nitrogen freezing.
+
-
Buffer I: 100mM RbCl 1.2g/100mL
+
Buffer I: 100mM RbCl 1.2g/100mL<br>
-
 45mM MnCl2·4H2O 0.891g/100mL
+
45mM MnCl2·4H2O 0.891g/100mL<br>
-
  30mM KAc (pH 7.5) 0.294g/100mL
+
30mM KAc (pH 7.5) 0.294g/100mL<br>
-
 10mM CaCl2·2H2O 0.147g/100mL
+
10mM CaCl2·2H2O 0.147g/100mL<br>
-
  glycerol (pH 6.8) 15% (w/v)
+
glycerol (pH 6.8) 15% (w/v)<br>
-
 
+
Buffer II: 10mM MOPS 0.209g/100mL<br>
-
Buffer II: 10mM MOPS 0.209g/100mL
+
10mM RbCl 0.12g/100mL<br>
-
  10mM RbCl 0.12g/100mL
+
75mM CaCl2·2H2O 1.1025g/100mL<br>
-
 75mM CaCl2·2H2O 1.1025g/100mL
+
glycerol (pH 6.8) 15% (w/v)
glycerol (pH 6.8) 15% (w/v)
</pre>             
</pre>             
Line 247: Line 236:
             <dt><a href="###">Measuring GFP expression with ELISA</a></dt>
             <dt><a href="###">Measuring GFP expression with ELISA</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br>
-
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
+
(2) Incubate at 37℃ for 20h with vigorous shaking.<br>
-
(2) Incubate at 37℃ for 20h with vigorous shaking.
+
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br>
-
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.
+
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm)   <br>      
-
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm)
+
-
</pre>          
+
             </dd>
             </dd>
         </dl>
         </dl>
Line 259: Line 246:
             <dt><a href="###">Observation GFP expression with Confocal Microscope</a></dt>
             <dt><a href="###">Observation GFP expression with Confocal Microscope</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br>
-
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
+
(2) Incubate at 37℃ for 20h with vigorous shaking.<br>
-
(2) Incubate at 37℃ for 20h with vigorous shaking.
+
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.<br>
-
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.
+
(4) Observation GFP expression with Confocal Microscope. <br>        
-
(4) Observation GFP expression with Confocal Microscope.
+
-
</pre>          
+
             </dd>
             </dd>
         </dl>
         </dl>
Line 271: Line 256:
             <dt><a href="###">Drawing growth curve</a></dt>
             <dt><a href="###">Drawing growth curve</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
 
-
(1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.
+
(1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.<br>
-
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.
+
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.<br>
-
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each.  
+
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each. <br>
-
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)
+
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)<br>
-
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.
+
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.<br>
-
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.
+
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.<br>
-
(7) Set the speed of the shaker to 200rpm.
+
(7) Set the speed of the shaker to 200rpm.<br>
-
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.
+
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.<br>
-
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)
+
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)<br>
-
(10) Record the data and analyze them.
+
(10) Record the data and analyze them. <br>        
-
</pre>          
+
             </dd>
             </dd>
         </dl>  
         </dl>  
Line 289: Line 273:
             <dt><a href="###">HPLC</a></dt>
             <dt><a href="###">HPLC</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.<br>
-
(1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.
+
(2) Dilute the mother solution with LB into 500μmol/L.<br>
-
(2) Dilute the mother solution with LB into 500μmol/L.
+
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.<br>
-
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.
+
(4) Add 10mL 500μmol/L atrazine solution into each tube.<br>
-
(4) Add 10mL 500μmol/L atrazine solution into each tube.
+
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.<br>
-
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.  
+
(6) Put all the tubes into the shaker, set the speed to 200rpm.<br>
-
(6) Put all the tubes into the shaker, set the speed to 200rpm.
+
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.<br>
-
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.
+
(8) After 24h of culture, take all the tubes out and put them on the ice.<br>
-
(8) After 24h of culture, take all the tubes out and put them on the ice.
+
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.<br>
-
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.
+
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.<br>
-
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.
+
(11) Analyze the samples with HPLC. <br>        
-
(11) Analyze the samples with HPLC.
+
-
</pre>          
+
             </dd>
             </dd>
         </dl>  
         </dl>  
Line 308: Line 290:
             <dt><a href="###">Verification of the motility from phenotype</a></dt>
             <dt><a href="###">Verification of the motility from phenotype</a></dt>
             <dd class="dd_1">
             <dd class="dd_1">
-
<pre>
+
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.<br>
-
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
+
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.<br>
-
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.
+
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.<br>
-
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.
+
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.<br>
-
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.
+
(5) Incubate at 37℃ for 20h.<br><br>
-
(5) Incubate at 37℃ for 20h.
+
(6) Take photos with Gel Analysis System.<br>
-
(6) Take photos with Gel Analysis System.
+
</pre>             
</pre>             
             </dd>
             </dd>

Revision as of 04:00, 24 September 2013

Extraction of mRNA
Kit
RT-PCR
Kit
PCR
(1) System: 2*PrimeStar 12.5µL Forward primer 1~2.5μL Reverse primer 1~2.5μL Bacteria solution 1~2μL dd water up to 25μL (2) Steps: Step 1 94℃ Step 2 94℃ Step 3 Annealing temperature Step 4 72℃ Step 5 go to Step 2, 25~35 cycles Step 6 72℃ Step 7 Pause at 4℃
PCR with DNA as Template
(1) System: 2×PrimeStar 12.5μL   Forward primer 1μL   Reverse primer 1μL   Template DNA 0.5μL   dd water up to 25μL (2) Steps: Step 1 94℃   Step 2 94℃   Step 3 Annealing temperature   Step 4 72℃   Step 5 go to Step 2, 25~35 cycles   Step 6 72 ℃   Step 7 Pause at 4℃
Double Digestion (40μL System)
1) Mix the following in a PCR tube: Enzyme A 2μL Enzyme B 2μL 10Xbuffer 2 or 4μL (depend on the buffer used) BSA 0 or 4μL (depend on the buffer used) DNA 20μL (insert DNA) or 10μl (vector DNA) dd water up to 40μL (2) Incubate at appropriate temperature (30 or 37℃, depend on the enzymes used) for 8~10h (shorter time if the enzymes used have star activity). (3) Pause at 4 ℃. (4) Gel running and purify the digestion product.
Agarose gel electrophoresis
(1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.
(2) Melt the mixture in a microwave until the solution becomes clear.
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.
(4) Pour the solution into the gel casting tray with appropriate comb.
(5) Let the gel cool until it is solid.
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.
(9) Run the gel at 135V for about 20min.
Gel purification with Axygen Gel Extraction Kit
(1) Excise the DNA fragment from the agarose gel with a clean shaver blade.
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.
(6) Apply the solution from the last step to the column.
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.
Ligation
(1) Test the concentration of the DNA samples.
(2) Pipet the following into a PCR tube:
Linearized vector DNA
Insert DNA
Solution I
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.
(4) Pause at 4℃.
Tranformatiom
(1) Get the competent cell from -80℃ and wait for its fusion on ice.
(2) Add the entire ligation product or 1μL plasmid into the tube.
(3) Mix and incubate on ice for 30min.
(4) Heat pulse at 42℃ for 90sec.
(5) Put back the tube on ice and incubate for 5min.
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics.
Mini-prep with Axygen Mini-prep Kit
(1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.
(3) Repeat the last step.
(4) Resuspend compact cells in 250μL buffer S1.
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.
(7) Centrifuge at 12000rpm for 10min.
(8) Apply the supernatant from the last step to the column.
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.
(12) Repeat the last step.
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.
Preparation of competent cells
(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.
(8) Store at -80℃ after liquid nitrogen freezing.
Buffer I: 100mM RbCl 1.2g/100mL
45mM MnCl2·4H2O 0.891g/100mL
30mM KAc (pH 7.5) 0.294g/100mL
10mM CaCl2·2H2O 0.147g/100mL
glycerol (pH 6.8) 15% (w/v)
Buffer II: 10mM MOPS 0.209g/100mL
10mM RbCl 0.12g/100mL
75mM CaCl2·2H2O 1.1025g/100mL
glycerol (pH 6.8) 15% (w/v)
Measuring GFP expression with ELISA
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
(2) Incubate at 37℃ for 20h with vigorous shaking.
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm)
Observation GFP expression with Confocal Microscope
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
(2) Incubate at 37℃ for 20h with vigorous shaking.
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.
(4) Observation GFP expression with Confocal Microscope.
Drawing growth curve
(1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each.
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.
(7) Set the speed of the shaker to 200rpm.
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)
(10) Record the data and analyze them.
HPLC
(1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.
(2) Dilute the mother solution with LB into 500μmol/L.
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.
(4) Add 10mL 500μmol/L atrazine solution into each tube.
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.
(6) Put all the tubes into the shaker, set the speed to 200rpm.
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.
(8) After 24h of culture, take all the tubes out and put them on the ice.
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.
(11) Analyze the samples with HPLC.
Verification of the motility from phenotype
(1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.
(5) Incubate at 37℃ for 20h.

(6) Take photos with Gel Analysis System.