Difference between revisions of "Team:BostonU/Protocols"

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       <li><a href="#Electrophoresis">Agarose Gel Electrophoresis</a></li>
 
       <li><a href="#Electrophoresis">Agarose Gel Electrophoresis</a></li>
 
       <li><a href="#CellStock">Cell Stock</a></li>
 
       <li><a href="#CellStock">Cell Stock</a></li>
−
       <li><a href="#ColonyPCR">Colony PCR</a></li>
+
       <li><a href="#Miniprep">Miniprep</a></li>
−
       <li><a href="#ColonyPCR">Colony PCR</a></li>
+
       <li><a href="#GelExtraction">Gel Extraction</a></li>
−
       <li><a href="#ColonyPCR">Colony PCR</a></li>
+
       <li><a href="#PCRCleanup">PCR Cleanup</a></li>
 +
      <li><a href="#Digestion">Digestion</a></li>
 +
      <li><a href="#Ligation">Ligation</a></li>
 +
      <li><a href="#Transformation">Transformation</a></li>
 +
      <li><a href="#LiquidCultures">Liquid Cultures</a></li>
 +
      <li><a href="#TestCuts">Test Cuts</a></li>
 +
      <li><a href="#Gibson">Gibson</a></li>
 +
      <li><a href="#Recombination">Recombination Reaction</a></li>
 +
      <li><a href="#CellFree">Cell-Free Reaction</a></li>
 +
      <li><a href="#MakingCF">Making Cell Free</a></li>
 
     </ul>
 
     </ul>
 
<div id="protocol-accordion"class="mainwrap">
 
<div id="protocol-accordion"class="mainwrap">

Revision as of 18:48, 28 October 2017

OUR TEAM

Below is an interactive list of the laboratory techniques employed by the team. Click to navigate to that protocol

KOD PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Add 18.5 uL of deionized water to PCR tube
  2. Add 1.5 µl of both the requisite Forward and Reverse Primers to PCR tube
  3. Add 1 ul of DNA template
  4. Add 27.5 µl of KOD Hot start Master Mix to PCR tube
  5. Centrifuge for 10 seconds to remove air bubbles
  6. Place in Thermocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kb

Colony PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube
  2. Add 17.5 µl of deionized water to another PCR tube
  3. Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube
  4. Add 2 ul of water containing cells with DNA template sample
  5. Add 27.5 µl of KOD Hot Start Master Mix
  6. Centrifuge for 10 seconds to remove air bubbles
  7. Place in Thermocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kb

Overlap Extension PCR

Materials

  • 5% DMSO
  • 2x KOD Hot Start Master Mix
  • PCR Tubes
  • Forward Primers(s) [10 uM]
  • Reverse Primer(s) [10 uM]
  • DNA Template
  • Deionized water

Procedure

  1. Add equimolar concentrations of each DNA sample to a PCR tube
  2. Add 1.5 uL of each Forward and Reverse Primer
  3. Add 27.5 uL of KOD Hot Start Master Mix
  4. Add deionized water until the total volume is 50 uL
  5. Place in Themocycler

Thermocycler Conditions

Number of Cycles From 20 - 40
Annealing Temperature Set temperature to the lowest primer melt temperature
Extension Time If target size is:
< 500 bp run 10 sec/kb
500-1000 bp run 15 sec/kb
1000-3000 bp run 20 sec/kb
< 3000 bp run 20 sec/kb

Making a 1% Agarose Gel

Materials

  • Agarose
  • 1x TAE Buffer
  • Ethidium Bromide
  • 250 mL Flask

Procedure

  1. Add 0.6 g of agarose to 250 mL flask
  2. Add 60 mL of 1x TAE buffer and mix by swirling the flask
  3. Microwave mixture for 60 seconds
  4. Add 3 uL of Ethidium Bromide and mix gently
  5. Pour into gel tray with the desired comb size and wait 30 minutes for it to solidify

Agarose Gel Electrophoresis

Materials

  • Agarose Gel
  • 6x Loading Dye
  • 2 log DNA ladder
  • Gel Box
  • 1x TAE Buffer

Procedure

  1. Add 6x loading dye to each sample
  2. Place agarose gel into gel box
  3. Fill gel box with 1x TAE buffer to fill line
  4. Load 2 log ladder and samples into wells
  5. Run gel at 135 V for 30-40 minutes

Cell Stock

Materials

  • Liquid Cell Culture
  • 50% Glycerol
  • Cryofreezer Tube

Procedure

  1. Add 600 ul of glycerol into cryotube
  2. Add 600 ul of culture to cryotube
  3. Mix gently by pipetting up and down
  4. Freeze and store at -80℃