IGEM:Harvard/2006/Lab Intro
From OpenWetWare
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
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).
- 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)
- 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.