Team:University of Chicago/Notebook

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==Individual Lab Notebook Pages==
==Individual Lab Notebook Pages==
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[[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br>
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* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]
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[[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br>
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* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]
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[[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br>
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* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]
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[[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br>
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* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]
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[[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br>
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* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]
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[[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br><br>
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* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]
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'''If you did it, record it'''<br><br>
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Remember: '''If you did it, record it'''<br>
== Resources ==
== Resources ==
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[http://www.freewebs.com/genehackers/ Team Website (External Link)]
[http://www.freewebs.com/genehackers/ Team Website (External Link)]
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== Recipes ==
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== Materials ==
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=== Media ===
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*[[Team:University_of_Chicago/Notebook/Media|Media]]
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==== LB Broth (1 L) ====
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*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]
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* 1 L dH20
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*[[Team:University_of_Chicago/Notebook/Gels|Gels]]
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* 10 g Bacto tryptone
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*[[Team:University_of_Chicago/Notebook/Plasmids|Plasmids]]
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* 5 g Bacto yeast extract
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* 5 g NaCl
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* 1 mL 2N NaOH
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* 12 g agar (optional)
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** 3 g agar + 250 mL broth per bottle
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* Autoclave
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Note: Add 3 mL 1 M CaCl2 before phage transductions
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==== PYE ====
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This is a '''general-purpose Caulobacter medium''' and is typically used for plates and starter cultures from the freezer and for colonies off plates. Plates are incubated at 30C. Caulobacter colonies typically take 2-3 days to appear.
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Per liter,
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* 2g bacto-peptone
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* 1g yeast extract
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* 200mg magnesium sulfate heptahydrate
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* 100mg calcium chloride dihydrate
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Use deionized or distilled water; occasionally problems arise with less pure sources.
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==== M13 Sweet ====
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This is our '''production medium'''.
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After autoclaving a liter of de-ionized water in a flask, add
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* 40mL 25X M13 concentrate
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* 10mL Hutner's Mineral Base Concentrate
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We sometimes add a little extra calcium chloride (100mM stock to a final concentration of 1mM) to the flasks to encourage aggregates.
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When the 1X M13 Sweet medium is assembled as indicated above, the final concentrations are:
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* 0.5% glucose
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* 0.15% glutamate
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* 2mM phosphate
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* 5 mM imidazole
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* 0.04% ammonium chloride
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* 1% Hutner's Mineral Salts
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==== 25X M13 concentrate ====
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Component stock solutions:
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* 125.0g glucose (or 137.5g glucose monohydrate) in a total volume of 600mL dH<sub>2</sub>O
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* 47.7g L-glutamic acid sodium salt (MW 187.13) in a total volume of 200mL dH<sub>2</sub>O
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* 8.5g imidazole in a total volume of 100mL dH<sub>2</sub>O
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* 3.4g KH<sub>2</sub>PO<sub>4</sub> in a total volume of 50mL dH<sub>2</sub>O
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* 10g ammonium chloride in a total volume of 50mL dH<sub>2</sub>O
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All component stocks should be made in separate bottles, autoclaved, and once they are cooled, combined into a single sterile bottle. If the components are mixed first and then autoclaved, the stock turns black. Store the stock at room temperature or at 4C. Don't worry if after a few weeks the 25X stock turns color from clear-yellow to yellow-brown.
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==== SOB Medium ====
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Used in growing bacteria for preparing chemically competent cells
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Ingredients
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* 0.5% (w/v) yeast extract
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* 2% (w/v) tryptone
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* 10 mM NaCl
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* 2.5 mM KCl
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* 20 mM MgSO4
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Per liter:
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* 5 g yeast extract
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* 20 g tryptone
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* 0.584 g NaCl
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* 0.186 g KCl
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* 2.4 g MgSO4
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Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.
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* SOB medium is also available dry premixed from Difco, 0443-17.
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* Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.
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==== SOC MediuProxy-Connection: keep-alive
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Cache-Control: max-age=0
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(per 100 ml) ====
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Note This medium should be prepared immediately before use.
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* Add 2 ml of filter-sterilized 20% (w/v) glucose or 1 ml of filter-sterilized 2 M glucose to 100 ml sterile SOB
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=== Buffers ===
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==== CCMB80 ====
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* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
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* 80 mM CaCl2.2H2O (11.8 g/L)
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* 20 mM MnCl2.4H2O (4.0 g/L)
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* 10 mM MgCl2.6H2O (2.0 g/L)
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* 10% glycerol (100 ml/L)
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* adjust pH DOWN to 6.4 with 0.1N HCl if necessary
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** adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
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* sterile filter and store at 4°C
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* slight dark precipitate appears not to affect its function
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*Note: Create 1M concentration stocks to make buffer prep easier in future.
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'''CCMB80: with aqueous stocks'''
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*10 mM KOAc pH 7.0: 10mL of 1M stock/L
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*80 mM CaCl2.2H2O: 80mL/L
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*20 mM MnCl2.4H2O: 20mL/L
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*10 mM MgCl2.6H2O: 10mL/L
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*10% glycerol: 100 ml/L
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==== Hutner's Mineral Base Concentrate ====
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It is not difficult, but follow directions carefully.
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* 20g nitrilotriacetic acid (NTA)
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* 54.5g magnesium sulfate heptahydrate
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* 6.67g calcium chloride dihydrate
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* 18.5mg ammonium molybdate tetrahydrate
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* 198mg iron sulfate monohydrate (or 323.7mg of the heptahydrate)
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* 100mL Metals-44
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TO BE CONTINUED
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==== 5x Ligation Adjustment Buffer ====
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* Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
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* KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)
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* CaCl2 400 mM (200 ml/l of a 2 M solution)
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* MnCl2 100 mM (100 ml/l of a 1 M solution)
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* Glycerol 46.8% (468 ml/liter)
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* pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)
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** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST)
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* water to 1 liter
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* autoclave or sterile filter
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* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
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* Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.
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=== DNA and protein gels ===
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==== Agar ====
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===== 0.8% =====
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# Weigh out .8g agarose
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# Put agarose in 500mL Erlenmeyer flask.
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# Add 100mL 1X TBE Buffer. Swirl.
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# Microwave at 100% power until the agarose starts to dissolve. Every 30-40 seconds, stop and swirl. Continue microwaving until solution boils and agarose is dissolved.
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# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed.
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# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.
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# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.
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===== 1.2% =====
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Same as above but use 1.2 g agarose instead of .8g
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====Coomassie Blue Stain/Destain Recipe====
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450 ml water<br>
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450 ml methanol<br>
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100 ml 100% (glacial) acetic acid<br><br>
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#Add 2 g per liter coomassie blue for stain.  Destain is exactly the same, except do not add coomassie blue.
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#To stain the gel, incubate it with gentle shaking for at least 30 minutes.  Pour the stain back into the bottle (you can reuse it).  Rinse out the residual stain with a small amount of destain solution, and then incubate with gentle shaking for ~1 hour or more.  To speed up destaining, you can put some folded KimWipes in the dish.  The dye will adsorb to these, which will remove it from solution.
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#After your gel is destained, you can rehydrate it in distilled water and then image it and/or place it on a piece of filter paper and dry it with a gel dryer.
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== Protocols ==
== Protocols ==
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=== Growing Cells ===
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*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]
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==== TOP10 Competent Cells ====
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*[[Team:University_of_Chicago/Notebook/DH5-alpha competent cells|DH5-alpha competent cells]]
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* Prechill plasticware and glassware
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*[[Team:University_of_Chicago/Notebook/Transformations|Transformations]]
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*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]
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===== Preparing seed stocks =====
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*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]
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# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
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*[http://www.jobinyvon.com/Raman/Tutorial-Intro Raman Spectroscopy Tutorial]
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#* room temperature works well
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*[http://www.afmuniversity.org/ AFM Tutorial]
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# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
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*[[Team: University_of_Chicago/Notebook/SDS-PAGE|SDS-PAGE]]
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#* room temperature works well
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*[[Media:Tyrosinase Cat Oxidase Activity.pdf | Simple Tyrosinase spectroscopy activity.]]
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# Add glycerol to 15%
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# Aliquot 1 ml samples to Nunc cryotubes
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# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
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#* This step may not be necessary
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# Place in -80°C freezer indefinitely.
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===== Preparing competent cells =====
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# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
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#* This takes approximately 16 hours.
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#* Controlling the temperature makes this a more reproducible process, but is not essential.
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#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule
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#* Aim for lower, not higher OD if you can't hit this mark
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# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
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#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
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#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer
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# Gently resuspend in 80 ml of ice cold CCMB80 buffer
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#* sometimes this is less than completely gentle. It still works.
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# Incubate on ice 20 minutes
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# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
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# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells.
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# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
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# Incubate on ice for 20 minutes
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# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates
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# Store at -80°C indefinitely.
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#* Flash freezing does not appear to be necessary
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# Test competence (see below)
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# Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
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===== Measurement of competence =====
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# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
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#* This is at 10 pg/_l or 10-5 _g/_l
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#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
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# Hold on ice 0.5 hours
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# Heat shock 60 sec at 42C
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# Add 250 _l SOC
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# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
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#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.  
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#* For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
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#* Ampicillin and kanamycin appear to do fine with 1 hour growth
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# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
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#* Good cells should yield around 100 - 400 colonies
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#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
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#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA
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===== Glycerol Stocks =====
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====== Materials ======
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* 40% glycerol solution
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* Cryogenic vials
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====== Method ======
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# Add 1 ml of 40% glycerol in H2O to a cryogenic vial.  
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# Add 1 ml sample from the culture of bacteria to be stored.
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# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.  
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#* Alternatively, pipet to mix.
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# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
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# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
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# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.
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====== Notes ======
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While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.
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Prof. Schonbaum's version
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# Grow a 3 ml culture overnight
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# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols
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# Mix by vortexing and let sit for a little bit.  Then put the tubes in the -80°C freezer.
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Latest revision as of 22:09, 26 August 2008

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