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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 ...
Ron Weiss
Wed Feb 10 15:48:08 EST 1999