IGEM:Harvard/2006/Lab Intro: Difference between revisions
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[[Transforming_chemically_competent_cells]] | [[Transforming_chemically_competent_cells]] | ||
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**http://openwetware.org/wiki/Miniprep/Qiagen_kit | **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) | *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 - | |||
*Digest | *[http://parts.mit.edu/r/parts/partsdb/pgroup.cgi?pgroup=Plasmid Construction plasmids] | ||
**[http://parts.mit.edu/registry/index.php/Part:pSB1A3 pSB1A3] - Amp | |||
**[http://parts.mit.edu/registry/index.php/Part:pSB1AC3 pSB1AC3] - Amp/Cm | |||
**[http://parts.mit.edu/registry/index.php/Part:pSB1AK3 pSB1AK3] - Amp/Kan | |||
**[http://parts.mit.edu/registry/index.php/Part:pSB1AT3 pSB1AT3] - Amp/Tet | |||
*Hydrate and transform pSB1AT3 into competent cells | |||
*Grow overnight 37°C | |||
==June 9== | |||
*[http://parts.mit.edu/r/parts/htdocs/Assembly/index.cgi BioBrick Assembly] | |||
*[http://parts.mit.edu/r/parts/htdocs/Assembly/standard_assembly.cgi Standard Assembly of 2 Part] | |||
'''Old pages''' | |||
*[http://biobricks.ai.mit.edu/Assembly/BB_Assembly.htm Biobrick Assembly] | |||
*[http://biobricks.ai.mit.edu/Assembly/Assy_Alternatives.htm Assembly alternatives] | |||
**[http://biobricks.ai.mit.edu/Assembly/Assy_3A.htm updated 3A] | |||
**[http://biobricks.ai.mit.edu/Assembly/Assy_Sel_Mix_CcdB.htm CcdB] | |||
**[http://biobricks.ai.mit.edu/Assembly/Assy_Sel_Mix_Split.htm 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) | |||
*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. | *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). | *Qiagen gel-extraction kit. Quantitate on nanodrop to determine concentration. (see http://openwetware.org/wiki/Wittrup:_Gel_extraction and Qiagen manual). | ||
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*Transformation (same as Tuesday). | *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. | *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 | **Alternatively, come in late and grow cultures overnight and then continue on Saturday |
Revision as of 13:02, 8 June 2006
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)
- 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.
1058 from 9 am to 1 pm on:
6/13, 6/15, and 6/16. On 6/14 room 1058 was already booked, I have
reserved room 1079.
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,
- 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