If we begin with certainties, we shall end in doubts; but if we begin with doubts,
and are patient in them, we shall end in certainties.
Francis Bacon

Day 1: DNA manipulation

Additional Reading

Overview of Experiment

This lab session focuses on several procedures commonly used in molecular biology: plasmid DNA isolation; restriction digests of DNA (one opens up a vector and the other generates an insert); phosphatase treatment of digested vector. Today you begin working with two BioBricks™:  BBa_R0040 and BBa_E0840. Visit the Registry of Standard Biological Parts for additional information about BioBricks™.


Notes on Molecular Biological Procedures

    General Guidelines
  1. Maintain a clean work area
  2. Use a fresh pipet tip for every transfer (tips should be DNase/RNase free)
  3. Wear gloves to prevent nuclease contamination (and to protect yourself!)
  4. Sterilize solids and liquids by autoclaving 20 minutes at 121°C at 15 psi
    Pipetting Small Volumes
  1. Before beginning the procedure, thaw all frozen reagents and mix well
  2. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
    (Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the microcentrifuge rotors)
  3. Touch only the very tip to the surface of the solution (i.e., do NOT submerge the pipet tip into the solution)
  4. Most enzyme stocks are in 50% glycerol; these solutions are quite viscous and liquid will stick to the outside of the pipet tip so touch only the surface
  2. ALWAYS balance the load in the centrifuge
  3. Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the rotors
  4. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
  5. Don't forget the cover for the rotor
  6. DO NOT SLAM THE LIDS! (this action breaks the latch mechanisms)

Plasmid DNA mini prep

We're using a Zyppy™ Plasmid Miniprep Kit (Catalog No. D4036, Zymo Research Corp., Orange, CA) to isolate plasmid DNA from 3 ml overnight (O/N) bacterial cultures of R0040 and E0840; each team will isolate DNA from each culture. PROTOCOL (adapted from the Zyppy™ Plasmid Miniprep Kit INSTRUCTION MANUAL):
  1. Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed (in a microcentrifuge); discard the supernatant (into small waste beaker)
  2. REPEAT step 1 (in the same tube).

    NOTE: typically you would save a small amount of culture to streak a plate and prepare a fresh overnight for a glycerol stock; bacterial cultures can be stored indefinitely at -80°C without significant loss of viability in media containing at least 15% glycerol (v/v).

  3. Add 600 µl nuclease-free water (NF H2O) to the cell pellet and resuspend completely by gently pipetting up and down
  4. Add 100 µl of 7X Lysis Buffer (Blue) and mix by inverting tube 6 times
  5. Add 350 µl COLD Neutralization Buffer (Yellow), containing 100 µg/ml RNaseA, and mix thoroughly by inverting tube
  6. Centrifuge at 16,000 x g for 4 minutes
  7. Transfer supernatant to a Zymo-Spin™ II column (avoid disturbing pellet!)
  8. Put column in a 2 ml collection tube and centrifuge for 15 seconds (maximum speed, press and hold the "short" button)
  9. Discard flow-through and put column back into the collection tube
  10. Add 200 µl Endo-Wash Buffer to column and centrifuge for 15 seconds (maximum speed, press and hold the "short" button)
  11. Add 400 µl Zyppy™ Wash Buffer (containing ethanol) to the column and centrifuge for 30 seconds (maximum speed)
  12. Discard flow-through and centrifuge at 16,000 x g for an additional 15 seconds to remove the last drop of wash from the column (residual ethanol will inhibit DNA elution)
  13. Transfer column into a sterile 1.5 ml tube
  14. Add 30 µl Zyppy™ Elution Buffer (10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA) directly to the column matrix and wait one minute (room temperature)
  15. Centrifuge 15 seconds (maximum speed, press and hold the "short" button) to elute plasmid DNA

Restriction enzyme digests of BioBrick™ plasmids

You perform a double digest on R0040 and E0840; you also set up an uncut control for R0040 plasmid. On lab day 2, you will gel purify the insert from E0840, ligate this fragment to R0040 vector that has been phosphatased and gel purified, and transform competent bacteria with the new construct.

Our restriction enzymes are from New England Biolabs (NEB): record the units/µl for each enzyme you use; enzyme concentrations and CutSmart™ Buffer can be found on NEB's web site (see links below).  You can use the Double Digest Finder tool at NEB to select optimal reaction conditions for any two NEB restriction enzymes; a compromise in digestion efficiency of one enzyme may be necessary for certain double digests.  Each team should set up the following reactions in 1.5 ml tubes:
  1. Put 10 µl plasmid DNA (mini prep) in one tube for the E0840 double digest and 10 µl plasmid DNA in one tube for the R0040 double digest; put 5 µl R0040 plasmid DNA in a 3rd tube for an uncut control (store the rest of your plasmid DNA at -20°C)
  2. Add 6 µl NF H2O to R0040 double digest; add 6 µl NF H2O to E0840 double digest; add 13 µl NF H2O to uncut control (for a final volume of 20 µl)
  3. Add 2 µl 10X NEB CutSmart™ Buffer (10X) to each tube
  4. Add
  5. Gently flick tube to mix and pulse spin
  6. Incubate at 37°C for at least 15 minutes ("dry" heat block)
  7. Pulse-spin all samples
  8. Incubate R0040 uncut and E0840 digest at 80°C (oven) for 20 minutes  
  9. Pulse spin heat-inactivated samples and store at -20°C (in a benchtop cooler) until day 2

Phosphatase treatment of vector DNA

Treating the digested vector DNA with phosphatase decreases recircularization by removing both of the 5' phosphates required for ligation so that only a molecule with a 5' phosphate at each end (untreated insert fragment) will be ligated.  Theoretically, R0040 should not be able to recircularize since two enzymes with "sticky ends" were used; however, if some of the DNA is cut at just one of the restriction sites, the vector can recircularize--digestion with SpeI and PstI only removes a small spacer between the sites so you cannot see a size difference between single cut vs double cut DNA on an agarose gel. We routinely treat linearized vectors with alkaline phosphatase to decrease the background in ligation/transformation procedures.
  1. Add 2 µl (1 unit/µl) Shrimp Alkaline Phosphatase (rSAP) (NEB, catalog # M0371S) to R0040 digest; gently mix and pulse spin sample
  2. Incubate 30-60 minutes at 37°C ("dry" heat block)
  3. Pulse spin and store at -20°C (in a benchtop cooler) until day 2
  4. Lab day 2: Heat inactivate at 80°C for 20 minutes (while agarose gel is cooling)

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 16 October 2014