Team:University of Chicago/Notebook

From 2008.igem.org

(Difference between revisions)
m (Individual Lab Notebook Pages)
(Protocols)
 
(28 intermediate revisions not shown)
Line 12: Line 12:
==Individual Lab Notebook Pages==
==Individual Lab Notebook Pages==
-
[[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]<br>
+
* [[Team:University_of_Chicago/Notebook/Damonwang|Damon Wang's notebook]]
-
[[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]<br>
+
* [[Team:University_of_Chicago/Notebook/Parijata |Parijata "Jata" notebook ]]
-
[[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]<br>
+
* [[Team:University_of_Chicago/Notebook/Norayucel|Nora Yucel's notebook]]
-
[[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]<br>
+
* [[Team:University_of_Chicago/Notebook/Lkstone|Laura Stone's notebook]]
-
[[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]<br>
+
* [[Team:University_of_Chicago/Notebook/Shorelandhall|Rob McConeghy's notebook]]
-
[[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]<br><br>
+
* [[Team:University_of_Chicago/Notebook/Dmchoi87|Daniel M. Choi's notebook]]
-
'''If you did it, record it'''<br><br>
+
 
 +
Remember: '''If you did it, record it'''<br>
== Resources ==
== Resources ==
Line 24: Line 25:
[http://www.freewebs.com/genehackers/ Team Website (External Link)]
[http://www.freewebs.com/genehackers/ Team Website (External Link)]
-
== Recipes ==
+
== Materials ==
-
=== Media ===
+
*[[Team:University_of_Chicago/Notebook/Media|Media]]
-
==== LB Broth (1 L) ====
+
*[[Team:University_of_Chicago/Notebook/Buffers|Buffers]]
-
* 1 L dH20
+
*[[Team:University_of_Chicago/Notebook/Gels|Gels]]
-
* 10 g Bacto tryptone
+
*[[Team:University_of_Chicago/Notebook/Plasmids|Plasmids]]
-
* 5 g Bacto yeast extract
+
-
* 5 g NaCl
+
-
* 1 mL 2N NaOH
+
-
* 12 g agar (optional)
+
-
** 3 g agar + 250 mL broth per bottle
+
-
* Autoclave
+
-
Note: Add 3 mL 1 M CaCl2 before phage transductions
+
-
 
+
-
==== SOB Medium ====
+
-
Used in growing bacteria for preparing chemically competent cells
+
-
 
+
-
Ingredients
+
-
* 0.5% (w/v) yeast extract
+
-
* 2% (w/v) tryptone
+
-
* 10 mM NaCl
+
-
* 2.5 mM KCl
+
-
* 20 mM MgSO4
+
-
 
+
-
Per liter:  
+
-
* 5 g yeast extract
+
-
* 20 g tryptone
+
-
* 0.584 g NaCl
+
-
* 0.186 g KCl
+
-
* 2.4 g MgSO4
+
-
 
+
-
Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.
+
-
 
+
-
* SOB medium is also available dry premixed from Difco, 0443-17.
+
-
* Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.
+
-
 
+
-
==== SOC Medium (per 100 ml) ====
+
-
Note This medium should be prepared immediately before use.
+
-
* Add 2 ml of filter-sterilized 20% (w/v) glucose or 1 ml of filter-sterilized 2 M glucose to 100 ml sterile SOB
+
-
 
+
-
=== Buffers ===
+
-
==== CCMB80 ====
+
-
* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
+
-
* 80 mM CaCl2.2H2O (11.8 g/L)
+
-
* 20 mM MnCl2.4H2O (4.0 g/L)
+
-
* 10 mM MgCl2.6H2O (2.0 g/L)
+
-
* 10% glycerol (100 ml/L)
+
-
* adjust pH DOWN to 6.4 with 0.1N HCl if necessary
+
-
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
+
-
* sterile filter and store at 4°C
+
-
* slight dark precipitate appears not to affect its function
+
-
 
+
-
*Note: Create 1M concentration stocks to make buffer prep easier in future.
+
-
'''CCMB80: with aqueous stocks'''
+
-
*10 mM KOAc pH 7.0: 10mL of 1M stock/L
+
-
*80 mM CaCl2.2H2O: 80mL/L
+
-
*20 mM MnCl2.4H2O: 20mL/L
+
-
*10 mM MgCl2.6H2O: 10mL/L
+
-
*10% glycerol: 100 ml/L
+
-
 
+
-
==== 5x Ligation Adjustment Buffer ====
+
-
* Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
+
-
* KOAc 40 mM (40 ml/liter of 1 M KOAc soluProxy-Connection: keep-alive
+
-
Cache-Control: max-age=0
+
-
 
+
-
on, pH 7.0)
+
-
* CaCl2 400 mM (200 ml/l of a 2 M solution)
+
-
* MnCl2 100 mM (100 ml/l of a 1 M solution)
+
-
* Glycerol 46.8% (468 ml/liter)
+
-
* pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)
+
-
** 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)
+
-
* water to 1 liter
+
-
* autoclave or sterile filter
+
-
* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
+
-
* Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.
+
-
 
+
-
=== DNA and protein gels ===
+
-
==== Agar ====
+
-
===== 0.8% =====
+
-
# Weigh out .8g agarose
+
-
# Put agarose in 500mL Erlenmeyer flask.
+
-
# Add 100mL 1X TBE Buffer. Swirl.
+
-
# 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.
+
-
# After agarose is dissolved, cover flask with foil or aran wrap and place in 55-60C waterbath until needed.
+
-
# When Agarose has cooled to enough to touch comfortably, add 10microliters Syber Safe. Swirl.
+
-
# Pour 30mL gels. Use 50Ml conical tube to measure 30mL.
+
-
===== 1.2% =====
+
-
Same as above but use 1.2 g agarose instead of .8g
+
== Protocols ==
== Protocols ==
-
=== Growing Cells ===
+
*[[Team:University_of_Chicago/Notebook/TOP10 competent cells|TOP10 competent cells]]
-
==== TOP10 Competent Cells ====
+
*[[Team:University_of_Chicago/Notebook/DH5-alpha competent cells|DH5-alpha competent cells]]
-
* Prechill plasticware and glassware
+
*[[Team:University_of_Chicago/Notebook/Transformations|Transformations]]
-
 
+
*[[Team:University_of_Chicago/Notebook/Glycerol stocks|Glycerol stocks]]
-
===== Preparing seed stocks =====
+
*[[Team:University_of_Chicago/Notebook/DNA mini-prep|DNA mini-prep]]
-
# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
+
*[http://www.jobinyvon.com/Raman/Tutorial-Intro Raman Spectroscopy Tutorial]
-
#* room temperature works well
+
*[http://www.afmuniversity.org/ AFM Tutorial]
-
# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
+
*[[Team: University_of_Chicago/Notebook/SDS-PAGE|SDS-PAGE]]
-
#* room temperature works well
+
*[[Media:Tyrosinase Cat Oxidase Activity.pdf | Simple Tyrosinase spectroscopy activity.]]
-
# Add glycerol to 15%
+
-
# Aliquot 1 ml samples to Nunc cryotubes
+
-
# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
+
-
#* This step may not be necessary
+
-
# Place in -80°C freezer indefinitely.
+
-
 
+
-
===== Preparing competent cells =====
+
-
# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
+
-
#* This takes approximately 16 hours.
+
-
#* Controlling the temperature makes this a more reproducible process, but is not essential.
+
-
#* Room temperature will work. You can adjust this temperature somewhat to fit your schedule
+
-
#* Aim for lower, not higher OD if you can't hit this mark
+
-
# Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
+
-
#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
+
-
#* It is often easier to resuspend pellets by mixing before adding large amounts of buffer
+
-
# Gently resuspend in 80 ml of ice cold CCMB80 buffer
+
-
#* sometimes this is less than completely gentle. It still works.
+
-
# Incubate on ice 20 minutes
+
-
# Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
+
-
# Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells.
+
-
# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
+
-
# Incubate on ice for 20 minutes
+
-
# Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates
+
-
# Store at -80°C indefinitely.
+
-
#* Flash freezing does not appear to be necessary
+
-
# Test competence (see below)
+
-
# 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.
+
-
 
+
-
===== Measurement of competence =====
+
-
# Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
+
-
#* This is at 10 pg/_l or 10-5 _g/_l
+
-
#* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
+
-
# Hold on ice 0.5 hours
+
-
# Heat shock 60 sec at 42C
+
-
# Add 250 _l SOC
+
-
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
+
-
#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.  
+
-
#* For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
+
-
#* Ampicillin and kanamycin appear to do fine with 1 hour growth
+
-
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
+
-
#* Good cells should yield around 100 - 400 colonies
+
-
#* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
+
-
#* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA
+
-
 
+
-
===== Glycerol Stocks =====
+
-
 
+
-
====== Materials ======
+
-
* 40% glycerol solution
+
-
* Cryogenic vials
+
-
 
+
-
====== Method ======
+
-
# Add 1 ml of 40% glycerol in H2O to a cryogenic vial.  
+
-
# Add 1 ml sample from the culture of bacteria to be stored.
+
-
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.  
+
-
#* Alternatively, pipet to mix.
+
-
# Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
+
-
# On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
+
-
# Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.
+
-
 
+
-
====== Notes ======
+
-
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.
+
-
Prof. Schonbaum's version
+
-
# Grow a 3 ml culture overnight
+
-
# Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols
+
-
# Mix by vortexing and let sit for a little bit.  Then put the tubes in the -80°C freezer.
+

Latest revision as of 22:09, 26 August 2008

Flatheader.png
Home Team Project Parts Modeling Notebook Meetings Papers


Contents

Lab Calendar

Click to see what lab work was done each day!


Individual Lab Notebook Pages

Remember: If you did it, record it

Resources

[http://www.freewebs.com/genehackers/ Team Website (External Link)]

Materials

Protocols