IGEM:Harvard/2006/Lab Intro

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June 5

Parts:

R0010 - LacI promoter

  • Plate 1, Well 7K
  • amp resistance
  • Hydrated yellow

E0241 - GFP reporter

  • Plate 2, Well 15L
  • amp resistance
  • Hydrated red

E7104 E7104 - GFP reporter behind T7 promoter

  • Plate 2, Well 13F
  • amp resistance
  • Hydrated red
  • Digest R0010 - SpeI + PstI (prefix)
  • Digest E0241 - XbaI + PstI (Suffix)

Standard Assembly Information Assembly Overview

Restriction Enzymes Used

Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing

Biobrick Delivery

Transforming_chemically_competent_cells

June 6

  • pick colonies and inoculate overnight cultures


June 7

  • Remove plates from 37C
  • Pick 3-5 colonies from each plate, plus 1 spot away from any colony as a control. I usually use a pipette tip, and just pop the whole thing into a culture tube containing 2 ml LB media (+ antibiotic).
  • Grow on shaker at 37C overnight

June 8

  • Pour a gel, and let this cool during step 5.
    • We can run a 1% gel.
    • Weigh out 1 g agarose and add 1x TBE to a total of ~100 g.
    • Squirt in a few extra grams of distilled water to allow for evaporation.
    • Microwave until agarose is fully dissolved (try 1 minute, followed by 30 second increments.
  • Qiagen miniprep kit on all samples.
  • Make glycerol stocks at this point. (Add 50 ul cells + 50 ul 30% glycerol to 1.5 ml tube, mix by pipetting and place in -80 freezer)
  • Hydrate and transform pSB1AT3 into competent cells
  • Grow overnight 37°C

June 9

Old pages

  • miniprep pSB1AT3
    • Digest - E + P (construction)
  • R0010
    • Digest - E + S (part 1: prefix)
    • plasmid pSB1A2 (amp resistance)
  • E0241
    • Digest - X + P (part 2: suffix)
    • plasmid: pSB1A2 (amp resistance)



  • Run samples on gel, cut out bands of proper size. Make sure to weigh empty tubes beforehand, so we will know the weight of the band.
  • Qiagen gel-extraction kit. Quantitate on nanodrop to determine concentration. (see http://openwetware.org/wiki/Wittrup:_Gel_extraction and Qiagen manual).
  • (pour a gel at this point)
  • Ligation (follow protocol that comes with T4 ligase, for example), with proper controls. While ligation runs, figure out what size products you are expecting.
  • Gel purification of ligated product. Same as steps 4-7.
  • Transformation (same as Tuesday).
  • Come in early (e.g. 8 am) and pick colonies so we can miniprep them the same day. Grow at 37C for 6-8 hours.
    • Alternatively, come in late and grow cultures overnight and then continue on Saturday
  • Miniprep & nanodrop quantitation
  • Gel analysis / purification
  • Submit samples for sequencing.

June 12

  • 10:00 - lectures + lunch (room 1058)
  • 12:30 - tour, etiquette, wiki notebooks
  • 12:45 - quick intro to folding DNA, prepare folding rxns and start folding, pour gels
  • 13:30 - quick intro to cloning, transformations, PCR, etc
  • 14:00 - transform, plate out bacteria.
  • 16:00 - load and run gels for folding reactions
  • 17:00 - image gels, plan for tomorrow

June 13

  • Room 1058 is booked from 9 am to 1 pm today
  • 10:00 - pick colonies, start cultures
  • 10:45 - present previous project (1 per student)
  • 14:00 - discussion
  • 16:00 - minipreps

June 14

  • Room 1079 is booked from 9 am to 1 pm today
  • 10:00 - digest, ligate, gel purify, transform DNA
  • 16:00 - discussion

June 15

  • Room 1058 is booked from 9 am to 1 pm today
  • minipreps, digests, run gels, submit samples for sequencing, phenotypic assays, discussion

June 16

  • Room 1058 is booked from 9 am to 1 pm today
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