Team:University of Chicago/Notebook

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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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* Prechill plasticware and glassware
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===== Preparing seed stocks =====
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# Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
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#* room temperature works well
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# Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
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#* room temperature works well
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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, pSProxy-Connection: keep-alive
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AT3) which are chloramphProxy-CProxyProxy-Connection: keep-alive
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onneProxy-Connection: keep-alive
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ion: keep-alive
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nection: keep-alive
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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 =====
===== Glycerol Stocks =====
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#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture.  The procedure should be modified as follows:  '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed.  '''1B)''' Discard the supernatant.  '''1C)''' Repeat steps 1A and 1B as needed.  '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely.  
#*The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture.  The procedure should be modified as follows:  '''1A)''' Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed.  '''1B)''' Discard the supernatant.  '''1C)''' Repeat steps 1A and 1B as needed.  '''1D)''' Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely.  
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times.  Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.
# Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times.  Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.
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# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly.  The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form.  Invert the sample an additional 2-3 times to ensure complete neutralization.
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# Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly.  The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form.  Invert the sample an additional 2-3 times to ensure complete Proxy-Connection: keep-alive
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utralization.
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.  
#Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.  
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''
# Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.'''Avoid disturbing the cell debris pellet. '''

Revision as of 18:46, 29 July 2008

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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

To see what each of us has done each day, go to individual notebook pages. And remember: If you did it, record it

Resources

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

Recipes

Protocols

Glycerol Stocks
Materials
  • 40% glycerol solution
  • Cryogenic vials
Method
  1. Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
  2. Add 1 ml sample from the culture of bacteria to be stored.
  3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
    • Alternatively, pipet to mix.
  4. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
  5. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
  6. 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

  1. Grow a 3 ml culture overnight
  2. Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols
  3. Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.

DNA prep

Zyppy mini-prep

The following procedure is performed at room temperature. Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion).

  1. Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube
    • The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: 1A) Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. 1B) Discard the supernatant. 1C) Repeat steps 1A and 1B as needed. 1D) Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely.
  2. Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.
  3. Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete Proxy-Connection: keep-alive

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utralization.

  1. Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.
  2. Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.Avoid disturbing the cell debris pellet.
  3. Place the column into a Collection Tube and centrifuge for 15 seconds.
  4. Discard the flow-through and place the column back into the same Collection Tube.
  5. Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube.
  6. Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds.
  7. Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature.
  8. Centrifuge for 15 seconds to elute the plasmid DNA.

Notes

  1. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been

neutralized and mixed thoroughly.

  1. Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer.
  2. The ZyppProxy-Connection: keep-alive

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�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral pH) can also be used to elute the DNA.

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