NanoBio: Plasmid Verification
Jump to navigationJump to search
Growing up more plasmid DNA
- Touch a sterile plastic pipet tip to chosen colony (only one colony!).
- Dip then swish tip in 5 mL LB media containing 5 µL Amp (100 mg/mL in distilled water) in a 14 mL tube.
- Shake the tube at 37 °C for 12-16 hrs.
- Use Qiagen's Spin Mini-prep kit to mini-prep the vector from the culture. Follow the instructions provided, except elute with 30 µL rather than 50 µL.
- The typical yield from a 5 mL culture is ~10 µg for the pSB1AC3, pSB1AK3, & pSB1AT3 plasmids.
Analytical Digestion and Gel of Biobricks Part
- Before doing a digest, check that the size of your new part is significantly different by ~20% or 500 bp (whichever is smaller) from the vector backbone and any of the component inserts you used to construct it.
- If the insert is greater than 100 bp in length, then you can digest with any combination of prefix (EcoRI, XbaI) and suffix (SpeI, PstI) enzymes. My default digestion is X|P.
- If the insert is less than 100 bp, try to find a non-Biobrick/Biofusion enzyme that will cut inside your insert. Alternatively, digest with ApaLI and one of the Biobrick/Biofusion enzymes so that your insert is within a fragment of ~550 bp.
- 2 µL mini-prepped DNA (~200 ng DNA)
- 1 µL 10x FastDigest Buffer
- 0.5 µL FastDigest XbaI (only 0.5 units are required)
- 0.5 µL FastDigest PstI (only 0.5 units are required)
- distilled water to bring to 10 µL total volume
- Run a 1-2% agarose gel to check for the presence and size of the insert.
Sequencing of Biobricks or Biofusion Part
- Only sequence new parts, final devices, and useful intermediates.
- Sequencing provides ~1000 bp of information.
- Confirm the entire sequence of the BioBricks part. This may require sequencing using both forward & reverse primers.
- For each sequencing reaction, submit ~ 500-2000 ng of plasmid DNA and 3.2 pmol of one appropriate primer in a total volume of 13 uL.
- In a 1.5 mL eppendorf, mix:
- plasmid DNA (I shoot for ~500 ng)
- 6.5 uL 2x primer solution
- water to a total volume of 13 uL.
- 2x primer solution = 3.2 pmol primer/6.5 uL solution, or 4.9 uL 100 mM primer plus 995 uL sterile water.
- To submit your requests, fill out and print the order form. Drop your tubes off at Stanley Hall Rm 237.
Archiving the New Part
- All new parts should be stored in the Molecular Foundry strain collection; these are the blue labeled boxes on the Common Stocks shelf in the -80C in Rm 5204. It is also helpful to keep parts that you have made in your personal strain collection.
- For the plasmid collection, place 5 uL mini-prepped plasmid DNA in a labeled eppendorf tube in the appropriate plasmid collection freezer box in the -20 freezer in Sm920.
- For the strain collection, grow a bacterial culture either to mid-exponential phase or overnight. Mix equal volumes 50% glycerol and culture in an autoclaved 2 mL screw-top tube. Store at -80.