Berkeley Structural Genomics Center
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BSGC PROTOCOLS AND METHODS  
   

Here are descriptions of our protocols and methods, sorted according to the steps in our experimental pipeline.

Cloning:

  • Manual LIC
    • LIC Primer Design
      This protocol describes the procedures needed to design the primers for amplification of the inserts that will be cloned into the BSGC LIC vectors (pB1-pB7).
      [More Information (PDF)]

    • LIC Vector Prep
      This protocol describes the standard procedure for preparing LIC Vector Stocks. Qiagen Kit purified vector, 5 ug, is digested with SmaI in 50 ul NEB#4. Digestion checked using 1 ul on agarose gel electrophoresis. T4 DNA polymerase exonuclease digestion in the presence of dATP yields LIC sticky ends. Gel purification of vector is not essential if SmaI digestion is complete, however if background is high, it might be necessary.
      [More Information (PDF)]

    • LIC Insert Prep Manual
      This protocol describes the standard procedure for preparing LIC Insert Stocks. Qiagen Kit purified Target PCR, 1 ug, is digested with T4 DNA polymerase exonuclease digestion in the presence of dTTP yields LIC sticky ends.
      [More Information (PDF)]

    • LIC Transformation Manual
      This protocol describes the standard procedure for LIC reactions of prepared vector and Target Insert and transformation into competent TOP10 cells. The transformation is split between plating and liquid culture. The plating gives individual colonies for clone isolation. The liquid culture yields a "mixed" miniprep of plasmid for characterizaton of the reaction/transformation and potential isolation of insert containing vectors.
      [More Information (PDF)]

    • T4 Insert Prep Manual
      A microplate of normalized clean PCR product is treated to produce T4-treated inserts.
      [More Information (PDF)]

  • Robotic LIC
    • PCR Setup
      PCR setup using Biomek 2000.
      [More Information (PDF)]

    • PCR Normalization
      Input: Cleaned-up PCR product plate and quantitation data. Output: Normalized plate, 1 picomole in 50 ul water. Next method: T4 polymerase reaction.
      [More Information (PDF)]

    • PCR cleanup
      This procedure removes the PCR reagents from the PCR product, resulting in 100 ul of clean PCR product. The next procedure is Quantitation.
      [More Information (PDF)]

    • Insert Prep
      This protocol describes the standard procedure for preparing LIC Insert Stocks on the Biomek 2000. Purified Target PCR DNA is normalized and 0.4 pmol DNA is digested in a 40 ul reaction with T4 DNA polymerase exonuclease digestion in the presence of dTTP yields LIC sticky ends.
      [More Information (PDF)]

    • LIC Transform
      The LIC reaction inserts the Target gene into the vector plasmid. During the Transformation reaction, the plasmid is taken up by an E. coli cell, the cells multiply in the culture media, each forms a colony when plated on agar prepared with the antibiotic against which the plasmid confers resistance. See T4 Reaction protocol and Vector Preparation protocol.
      [More Information (PDF)]

    • E-gel Run
      This method was developed to analyze the PCR screen of plasmid clones, with columns of 8 clones per gene. The 10 ul PCR screen rxn is diluted up to 25 ul in the PCR plate, because 20 ul are needed in each well of the E-gel. Empty wells must be loaded with 20 ul of water. The DNA standard is loaded manually. The Biomek loads the samples on the E-gel on the E-gel Motherbase, which is then plugged in for the electrophoresis. A U.V.digital picture is taken of the gel and saved on a disk for transfer to a computer for editing and analysis.
      [More Information (PDF)]

Expression:

Cell Paste Preparation:

  • Inoculum for Paste SOP
    Inoculum for Maxi Growth of Membrane Proteins.
    [More Information (PDF)]

  • Studier New Protocol for Auto Inducing Media
    Studier Method for Induction, based on Bill Studier's protocol.
    [More Information (PDF)]

Purification:

  • AKTA 3D SOP
    Protocol for purifying proteins via the 3DEXPII method (affinity, desalt, ion exchange) on the AKTA.
    [More Information (PDF)]

  • AKTA Ion Exchange (IEX) Chromatography SOP
    Protocol for purifying proteins via IEX on the AKTA.
    [More Information (PDF)]

  • AKTA Metal Chelating (MC) Chromatography SOP
    Protocol for purifying proteins via MC on the AKTA.
    [More Information (PDF)]

  • HisTrap HP, Ni Sepharose media
    Ni Sepharose media and His Trap HP pre-packed columns utilize a new ligand chemistry to chelate metal ions for metal affinity chromatography. This media has a higher binding capacity and tolerates a wider range of chemical conditions than HiTrap MC media. HisTrap HP media and columns come pre-charged with Ni. It is recommended to strip and recharge the media/ columns between use with different targets. The column can be used up to 6 times with the same target.
    [More Information (PDF)]

  • mTEV Digestion
    Protocol for cleaving proteins with mTEV protease.
    [More Information (PDF)]

Protein Characterization:

  • Native PhastGel Electrophoresis SOP
    The purpose of the Native PAGE technique is to determine the homogeneity of a protein according to its banding pattern on the gel.
    [More Information (PDF)]

  • Protein Concentration by UV280
    [More Information (PDF)]

  • Western Blot SOP
    Protocol to detect whether his tagged protein is present in a protein gel by transferring the protein bands to a nitrocellulose membrane and probing by using anti-his antibody.
    [More Information (PDF)]

Crystallization:

Crystallography:

  • Data Collection Strategy
    This flowchart shows how our crystallographers make decisions related to data collection.
    [More Information (PDF)]

  • Recommended flash-cooling and annealing procedures
    This flowchart shows our protocol for flash-cooling and annealing crystals.
    [More Information (PDF)]

  • Heavy atom soaking experiment
    This flowchart shows our protocol for heavy atom soaking.
    [More Information (PDF)]

NMR:

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