IGEM:Harvard/2006/Lab Intro

Parts:

R0010 - LacI promoter

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

E0241 - GFP reporter

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

E7104 - GFP reporter behind T7 promoter

• Plate 2, Well 13F
• amp resistance
• Hydrated red

• Digest R0010 - SpeI + PstI (prefix)
• Digest E0241 - XbaI + PstI (Suffix)

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

Biobrick Delivery

Transforming_chemically_competent_cells

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).
  • (see http://openwetware.org/wiki/Bacterial_cell_culture)
• 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.
  • http://openwetware.org/wiki/Miniprep/Qiagen_kit
• 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)
• Digest R0010 - SpeI + PstI (prefix)
• Digest E0241 - XbaI + PstI (Suffix)
• 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).

June 9

• 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
• 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,
• 14:00 - transform, plate out bacteria.
• 16:00 - load and run gels for folding reactions
• 17:00 - image gels, plan for tomorrow

June 13

• 10:00 - pick colonies, start cultures
• 10:45 - present previous project (1 per student)
• 14:00 - discussion
• 16:00 - minipreps

June 14

• 10:00 - digest, ligate, gel purify, transform DNA
• 16:00 - discussion

June 15

• minipreps, digests, run gels, submit samples for sequencing, phenotypic assays, discussion