IGEM:Harvard/2006/Lab Intro: Difference between revisions

Revision as of 10:22, 8 June 2006

Parts:

R0010 - LacI promoter

E0241 - GFP reporter

E7104 - GFP reporter behind T7 promoter

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

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

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).

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.

@@ Line 41: / Line 41: @@
 **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).