in vivo Experiments for Measuring Inverter Transfer Curves and a Ring Oscillator

The Bio Group
{bio,us}@ai.mit.edu
MIT Artificial Intelligence

 
• Contents
• List of Figures
• Time Sheet
• General Purpose Experiments
  • Grow Stock of pGFP(LVA) and pBR322
    • Inoculate  ,  , and  
    • Maxiprep   and   to Extract Plasmids
    • Estimate pBR322 and pGFP(LVA) DNA Concentrations from Prep
    • Analytical Gel on pBR322 and pGFP(LVA) Samples
    • Inoculate    again (100 ml)
    • Maxiprep   to Extract Plasmids
  • Prepare   for Electroporation
• Quantitate the Constitutive Expression Level of GFP fused to pBR322 P1 Promoter
  • Extract GFP(LVA) insert
    • Plate and inoculate BL21+pGFP(LVA)
    • Transform    with pGFP(LVA)
    • Miniprep    to extract plasmid
    • Restriction digest of pGFP(LVA) with HindIII
    • Separate digested GFP(LVA) insert with QIAEX agarose gel extraction kit
    • Second Attempt: Restriction Digest of pGFP(LVA)
    • Second Attempt: Separate digested GFP(LVA) insert with QIAEX agarose gel extraction kit
  • Create additional pBR322 vector
    • Inoculate and Grow  
    • Transform    with pBR322, plate, and grow
    • Inoculate    into LB Amp liquid and grow
    • Miniprep    to extract plasmid
    • Restriction digest of pBR322 with EcoRI
    • Analytical Gel to Check pBR322 digest
    • Second Attempt: Restriction Digest of pBR322
    • Second Attempt: Separate digested pBR322 insert with QIAEX agarose gel extraction kit
  • Create EcoRI to HindIII adapters
  • Transform and grow colonies of  
    • Ligation of GFP(LVA), pBR322, and HindIII-EcoRI Adapters
    • Electroporate pBR322+GFP(LVA) into  , and Inoculate
• Second Attempt: Quantitate the Constitutive Expression Level of GFP fused to pBR322 P1 Promoter
  • Create pBR322+GFP(LVA)
    • Digest pBR322 and pGFP(LVA) with 2 Enzymes Each
    • Gel Purify Digested pBR322 and GFP(LVA)
    • Anneal XbaI-EcoRI Adapter
    • Ligate pBR322, GFP(LVA), adapters
    • Gel Purify Ligation of pBR322 + GFP(LVA)
  • Quantitate expression levels of GFP on individual cells using FACS
    • Obtain calibration curve with fluorescent beads
    • Stain cells to enable distinguishing cells from debris
    • Run stained cells through cytometer and gather data
    • Analyze data
• Analyze Cistronic Linearity of GFP Expression Levels
  • put pGBM7 into  
• Generate and Measure Random Drives
• Protocol: Electroporation
  • Cell Preparation
  • Electroporation Procedure
• Protocol: Prep to Extract Plasmid (QIAGEN Prep Kits 20,100,500)
  • Qiagen-tip 20 Miniprep
  • Qiagen-tip 100 Midiprep or Qiagen-tip 500 Maxiprep
• Protocol: Making Plates
  • LB Amp plates
• Protocol: Making Gels
  • Agarose Gel
• Protocol: Gel Electrophoresis
  • Agarose Gel Electrophoresis
  • Restain Agarose Gel
• Protocol: Prepative Gel to Seperate and Extract DNA
  • Agarose Gel Extraction Protocol (QIAEX II)
• Concentrate DNA using Microcon Centrifugal Filtration
• About this document ...