Molecular Techniques and Methods

Quick Screening of Bacterial Transformants by PCR


Copy Right © 2001/ Institute of Molecular Development LLC


INTRODUCTION



MATERIALS AND SOLUTIONS

0.2% Tween-20 (10 ml)
0.2% Tween-20 ------------------------ 200 ul of 10% Tween-20
DW ------------------------------------- 10 ml



PROCEDURES

1. Pick a single colony transformant and grow in 2 ml LB with antibiotics for at least 4hrs.

  • Pick 8-10 colonies per gene.

    2. Once cell culture turbidity reaches A600 > 0.4, collect 1 ml culture in a microfuge tube.
  • Cell culture turbidity is determined by visually.
  • Save the rest of culture for later use.

    3. Centrifuge for 30 sec. at 12,000 rpm.

    4. Discard supernatant.

    5. Resuspend the cell pellet in 100 ul of 0.2% Tween-20 completely by vortexing.
  • If cell pellet is small, use 50 ul, 0.2% Tween-20.

    6. Heat cell suspension at 95oC for 5 min to disrupt cells.

    7. Centrifuge for 5 min. at 12,000 rpm.

    8. Use 2 ul of supernatant for PCR reaction.


    NOTES


    KIT INFORMATION


    REFERENCES


    Please send your comment on this protocol to "editor@MolecularInfo.com".

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