This is a cached page for the URL (http://www.laboratoryexperiments.com/LAB1.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
The Molecular Biology Labs - LAB 1
LISTS | LAB 2 | LAB 3 | LAB 4 | LAB 5 | LAB 6 | LAB 7 | LAB 8 | LAB 9 | APPEND 1 | APPEND 2

LaboratoryExperiments.com


The Molecular Biology Labs



LAB 1: Plasmid DNA Isolation from Bacteria

| MATERIALS | SUPPLIES | PROCEDURES | RESULTS |

Materials:

  1. TENS solution:
    • 10 mM Tris (pH to 7.5)
    • 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)
    • 0.1 N sodium hydroxide
    • 0.5 % sodium dodecyl sulfate
  2. 3 M Sodium acetate, pH 5.2
  3. Pre-chilled (at -20 degrees C) 100 % ethanol
  4. 70 % Ethanol
  5. Distilled water
  6. Overnight bacterial culture (LAB 5)
Supplies:
  1. Micropipetter and tips
  2. Vortex mixer
  3. Microcentrifuge and tubes
Procedures:
  1. Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge. (observation: bacteria form a pellet at the bottom of the tube.)
  2. Decant supernatant, leaving 50-100 ul in the tube.Teacher's note:  In order to resuspend cells completely, some supernatant should be   left, but too much will dilute out the TENS solution.
  3. Vortex to resuspend the bacteria pellet completely.
  4. Add 300 ul of TENS solution.
  5. Vortex for 5 seconds to mix. (observation: the contents of the tube should become slimy.)Teacher's note:  If more than 10 minutes are needed before moving on to the next step,   the tube should be set on ice/ice water bath to prevent degradation of bacterial chromosomal   DNA which can then coprecipitate with plasmid DNA.
  6. Add 150 ul of the sodium acetate.
  7. Vortex for 5 seconds to mix.
  8. Spin for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris, is formed at the bottom of the tube.)
  9. Transfer supernatant to a fresh tube.
  10. Add 0.9 ml of pre-chilled 100 % ethanol.
  11. Spin for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.)
  12. Discard supernatant and add 1 ml of 70 % ethanol.
  13. Discard the ethanol and add another 1 ml of 70 % ethanol.
  14. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
  15. Resuspend the pellet in 30 ul of distilled water and keep at 4 degrees C or -20 degrees C.Teacher's note:  DNA should be stored at   -20 degrees C, but repeated freeze-thaw should be avoided.  So if it is needed frequently or   in a couple of days, store it at 4 degrees C.
Results:
  1. Plasmid DNA is now ready for estimation of DNA concentration (LAB 4) followed by restriction digest (LAB 3).
  2. Typical yield is 2-3 ug.

| GO BACK TO TOP OF THIS PAGE |

This site is produced by Scio Corporation
Send comments or suggestions to contents@LaboratoryExperiments.com