IGEM:Harvard/2006/Lab Intro

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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

Current revision

Contents

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

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).
  • 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

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
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