IGEM:Harvard/2006/Lab Intro

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


 * About XbaI Origins of XbaI


 * About SpeI Origins of SpeI


 * EcoRI More info about EcoRI


 * PstI Origins of Pst1

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).
 * (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)


 * Construction plasmids
 * pSB1A3 - Amp
 * pSB1AC3 - Amp/Cm
 * pSB1AK3 - Amp/Kan
 * pSB1AT3 - Amp/Tet


 * Hydrate and transform pSB1AT3 into competent cells
 * Grow overnight 37°C

June 9

 * BioBrick Assembly
 * Standard Assembly of 2 Parts

Old pages
 * Biobrick Assembly
 * Assembly alternatives
 * updated 3A
 * CcdB
 * Split Gene Strategy


 * 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)


 * BioBrick Digests


 * 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

Folding DNA nanostructures

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