Team:NRP-UEA-Norwich/Notebook Protocols

From 2014.igem.org

(Difference between revisions)
Line 107: Line 107:
       </div>
       </div>
<div class="container">
<div class="container">
-
    <div id="accordion">
 
      
      
 +
    <div id="accordion">
     <h3 class="accordion-toggle"> GoldenGate Digestion-Ligation reaction (Level 1) </h3>
     <h3 class="accordion-toggle"> GoldenGate Digestion-Ligation reaction (Level 1) </h3>
     <div class="accordion-content">
     <div class="accordion-content">
Line 130: Line 130:
<h3 class="accordion-toggle"><i>E. coli</i> Calcium Chloride Heat Shock Transformation</h3>
<h3 class="accordion-toggle"><i>E. coli</i> Calcium Chloride Heat Shock Transformation</h3>
-
   
+
Aim: to get DNA expression in E. coli.</b><br><br>
-
<b> Aim: to get DNA expression in E. coli.</b><br><br>
+
<ul>
-
 
+
<li>Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.</li>
-
• Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.<br>
+
<li>Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.</li>
-
• Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.<br>
+
<li>Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.</li>
-
• Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.<br>
+
<li>Preheat water bath to 42oC.</li>
-
• Preheat water bath to 42oC.<br>
+
<li>Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).</li>
-
• Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).<br>
+
<li>Transfer back onto ice for 5 mins.</li>
-
• Transfer back onto ice for 5 mins.<br>
+
<li>Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.</li>
-
• Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.<br>
+
<li>Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.</li>
-
• Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.<br>
+
</ul>
 +
</div>
    
    
Line 179: Line 180:
<h3 class="accordion-toggle">Preparation for sequencing</h3>
<h3 class="accordion-toggle">Preparation for sequencing</h3>
-
<b>Aim: to sequence our DNA in order to check content of our constructs.</b><br><br>
+
Aim: to sequence our DNA in order to check content of our constructs.
-
• Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes. <br>
+
<ul>
-
• Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)).<br>
+
<li>Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes. </li>
-
• Send off both 10µl samples for sequencing.<br>
+
<li>Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl) </li>
 +
<li>Send off both 10µl samples for sequencing.</li>
 +
</ul>
 +
</div>

Revision as of 19:50, 16 October 2014

NRP UEA Norwich iGEM 2014

Lab Protocols

GoldenGate Digestion-Ligation reaction (Level 1)

PROTOCOL HERE

GoldenGate Digestion-Ligation reaction (Level 2)

PROTOCOL HERE

Making LB Agar

PROTOCOL HERE

Making LB broth

PROTOCOL HERE

E. coli Calcium Chloride Heat Shock Transformation

Aim: to get DNA expression in E. coli.

  • Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.
  • Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
  • Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
  • Preheat water bath to 42oC.
  • Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
  • Transfer back onto ice for 5 mins.
  • Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
  • Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.

E. coli Electroporation Transformation

PROTOCOL HERE

Making elctrocompetent Agrobacterium tumefaciens

PROTOCOL HERE

Agrobacterium tumefaciens Electroporation Transformation

PROTOCOL HERE

Blue- White Selection

PROTOCOL HERE

Colony PCR

PROTOCOL HERE

DNA mini-prep

PROTOCOL HERE

Preparation for sequencing

Aim: to sequence our DNA in order to check content of our constructs.
  • Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes.
  • Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
  • Send off both 10µl samples for sequencing.

Agrobacterium tumefaciens Infiltration

PROTOCOL HERE

Infiltration Analysis

PROTOCOL HERE

Making GoldenGate compatible PSB1C3 'Flipper'

PROTOCOL HERE

Antibiotic Selection

PROTOCOL HERE

Retrieved from "http://2014.igem.org/Team:NRP-UEA-Norwich/Notebook_Protocols"