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

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)

Old pages

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.

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

Room 1058 is booked from 9 am to 1 pm today
minipreps, digests, run gels, submit samples for sequencing, phenotypic assays, discussion

@@ Line 20: / Line 20: @@
 *Digest E0241 - XbaI + PstI (Suffix)
-Restriction Enzymes
+==Standard Assembly Information==
-*[http://rebase.neb.com/rebase/enz/XbaI.html About XbaI]
+[http://parts.mit.edu/wiki/index.php/Standard_Assembly Assembly Overview]
-[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=592365&dopt=Abstract XbaI source]
+<b>Restriction Enzymes Used</b>
-*[http://rebase.neb.com/rebase/enz/SpeI.html About SpeI]
+*[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://home1.gte.net/vsjslsk1/sphaerotilus.htm SpeI Source]
+*[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