Home

Additional Reading

• The second wave of synthetic biology, P. Purnick and R. Weiss (Nat Rev Mol Cell Biol 2009, 10(6):410-422)
• Life from information, C. Voigt (Nat Methods 2008, 5(1):27-28)
• Streamlined engineering for synthetic biology, J. Perkel (Nat Methods 2013,10:39-42)

Overview of Experiment

Background

• Sterile Technique
• DNA Mini Prep
• Restriction Enzymes

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

Centrifugation
1. DO NOT PUT TAPE ON TUBES!
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

Disposal of Bacterial Waste:
• Discard bacterial supernatant in a small beaker; add bleach to 10% for 10 minutes before dumping into the sewer
• Collect contaminated tips and culture tubes in a beaker at your desk then rinse the beaker with bleach after dumping the waste in the clear Biohazards bag; these bags will be autoclaved prior to placement in the household trash
• After the bacteria are lysed, tips, vials, and other materials should be discarded in the regular trash

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
   • Complete lysis is indicated when the solution changes from opaque to clear blue
   • You must proceed to step 5 within 2 minutes (excessive lysis can denature plasmid DNA!)
5. Add 350 µl COLD Neutralization Buffer (Yellow), containing 100 µg/ml RNaseA, and mix thoroughly by inverting tube
   • When neutralization is complete, sample turns yellow and a yellowish precipitate forms
   • Invert sample 2-3 more times (to ensure complete neutralization)
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

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
   • 1 µl SpeI-HF® (Catalog # R3133S) and 1 µl PstI-HF® (Catalog # R3140S) to R0040
   • 1 µl XbaI (Catalog # R0145S) and 1 µl PstI-HF® to E0840
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
   • R0040 digest: proceed to phosphatase treatment of vector DNA
   • R0040 uncut and E0840 digest: heat inactivate restriction enzymes as in step 8
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

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