NanoBio: Plasmid Verification

Growing up more plasmid DNA

 * 1) Touch a sterile plastic pipet tip to chosen colony (only one colony!).
 * 2) Dip then swish tip in 5 mL LB media containing 5 µL Amp (100 mg/mL in distilled water) in a 14 mL tube.
 * 3) Shake the tube at 37 °C for 12-16 hrs.
 * 4) 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.
 * 5) *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.
 * Mix:
 * 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.
 * For the pSB1AC3, pSB1AK3, and pSB1AT3 vector, use AF009 and  AF010  as forward and reverse primers.
 * For the V0100 or V0120 vectors, use AF039 &  AF040 as forward and reverse primers.
 * For any of the Sikorski vectors, use AF058 &  AF059 as forward and reverse primers.
 * 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.