IGEM:Harvard/2006/Lab Intro: Difference between revisions
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==June 5== | |||
Parts: | Parts: | ||
R0010 - LacI promoter | [http://parts.mit.edu/registry/index.php/Part:BBa_R0010 R0010] - LacI promoter | ||
* Plate 1, Well 7K | * Plate 1, Well 7K | ||
* amp resistance | * amp resistance | ||
* Hydrated yellow | * Hydrated yellow | ||
E0241 - GFP reporter | [http://parts.mit.edu/registry/index.php/Part:BBa_E0241 E0241] - GFP reporter | ||
* Plate 2, Well 15L | * Plate 2, Well 15L | ||
* amp resistance | * amp resistance | ||
* Hydrated red | * Hydrated red | ||
E7104 - GFP reporter behind T7 promoter | [http://parts.mit.edu/registry/index.php/Part:BBa_E7104 E7104] E7104 - GFP reporter behind T7 promoter | ||
* Plate 2, Well 13F | * Plate 2, Well 13F | ||
* amp resistance | * amp resistance | ||
Line 18: | Line 19: | ||
*Digest R0010 - SpeI + PstI (prefix) | *Digest R0010 - SpeI + PstI (prefix) | ||
*Digest E0241 - XbaI + PstI (Suffix) | *Digest E0241 - XbaI + PstI (Suffix) | ||
'''Standard Assembly Information''' | |||
[http://parts.mit.edu/wiki/index.php/Standard_Assembly Assembly Overview] | |||
<b>Restriction Enzymes Used</b> | |||
*[http://rebase.neb.com/rebase/enz/XbaI.html About XbaI] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=592365&dopt=Abstract Origins of XbaI] | |||
*[http://rebase.neb.com/rebase/enz/SpeI.html About SpeI] [http://home1.gte.net/vsjslsk1/sphaerotilus.htm Origins of SpeI] | |||
*[http://rebase.neb.com/rebase/enz/EcoRI.html EcoRI] [http://en.wikipedia.org/wiki/EcoRI More info about EcoRI] | |||
*[http://rebase.neb.com/rebase/enz/PstI.html PstI] [http://www.thelabrat.com/restriction/sources/Providenciastuartii.shtml Origins of Pst1] | |||
Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing | Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing | ||
[http://parts.mit.edu/wiki/index.php/Biobrick_delivery 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) | |||
*[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 Parts] | |||
'''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) | |||
*[[Knight:Restriction_Digest|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) | |||
*[[Knight:DNA_ligation_using_NEB_Quick_Ligation_Kit|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 | |||
[[IGEM:Harvard/2006/Folding_DNA_nanostructures|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 |
Latest revision as of 05:42, 14 June 2006
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
Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing
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).
- 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.
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
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