Team:BrownTwo/Protocols/compcells

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==Making Ultracompetent Cells==
==Making Ultracompetent Cells==
-
*Transformation can be a major roadblock in assembling DNA constructs.  Ligations can be hard to transform, especially with large inserts.  DNA blotted on paper by the registry tends to be even harder.  We have had much better luck with the following protocol from Inoue than the standard CaCl2 method:
+
*Transformation can be a major roadblock in assembling DNA constructs.  Ligations can be hard to transform, especially with large inserts.  DNA blotted on paper from the registry tends to be even harder.  We have had a great experience with the following protocol from Inoue, getting near lawns of transformants from ligations:
 +
 
 +
*Based on the OpenWetWare page: http://openwetware.org/wiki/Preparing_chemically_competent_cells_(Inoue)
 +
 
 +
*Makes about 25mL, or enough for nearly 500 50 uL transformations
==Materials==
==Materials==
-
* Plate of cells streaked for single colonies
+
* Plate of cells streaked for single colonies.  We used DH5alpha.
-
* [[SOB]]
+
* [http://openwetware.org/wiki/SOB SOB]
* Ice
* Ice
-
* [[TB buffer]]
+
* [http://openwetware.org/wiki/TB_buffer TB buffer]
-
* DMSO
+
* DMSO (99.9%)
-
* Dry Ice (or liquid nitrogen)
+
* Liquid nitrogen
==Glassware & equipment==
==Glassware & equipment==
-
* 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water)
+
* (3)1 liter Erlenmeyer flasks (no detergent residue- we rinsed well with pure water, then autoclaved 2/3 full of pure water, emptying just before use
-
* 220 ml conical centrifuge tubes BD 35 2075
+
* (1) 250 mL Erlenmeyer flask
-
* Eppendorf 5410R refrigerated centrifuge with conical adapters
+
* 250 mL centrifuge bottles, autoclaved
 +
* Refrigerated centrifuge
==Preparation==
==Preparation==
-
# Pick a 10 - 12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of sterile SOB medium (do not use LB) in a 2 liter flask.  Save some medium as an OD blank.
+
===Day 1===
-
# Grow to  an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important).  This is slow -- approximately 35-40 hours.
+
# Pour LB plates (no antibiotic)
-
# Prechill the centrifuge to 4 degrees
+
#  In the afternoon, streak a plate of DH5alpha from frozen stock.  (We usually just use a sterile 200 ul pipette tip, scrape some frozen cells off the top, melt them in a pool near an edge of the plate, return the still-frozen stock to the -80C freezer, and streak back and forth across the surface.)
-
# Remove from the incubator and place on ice for 10 minutes
+
#Incubate plate inverted at 37C overnight (14-20 hours)
-
# Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
+
# Make 1L of SOB medium in a bottle without detergent residue
-
# Drain the medium and resuspend each pellet first in 5 ml of ice cold TB.  Add  an additional 75 ml of cold TB buffer and resuspend.
+
# Rinse out the clean flasks very well to remove detergent residue- (3) 1L, (1) 250 mL.  Fill each 2/3 with pure water and autoclave. Keep the water in overnight.
-
# Place on ice for 10 minutes
+
-
# Spin down as above.
+
-
# While spinning, add 1.4 ml of DMSO to 18.6 ml of TB (7% DMSO mixture)
+
-
# Resuspend each pellet in 20 ml of cold TB-DMSO mixture
+
-
# Incubate on ice for 10 minutes
+
-
# Dispense cells into pre-chilled tubes
+
-
# Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
+
-
==Thoughts on improvements==
+
===Day 2===
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* "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
+
# At around 11 AM, pour the water out of the 250 mL flask and add 25mL SOB
-
* They also control pH at 7.5, which may be a major issue
+
# Innoculate with one colony from the LB plate
-
* Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
+
# Grow this starter culture at 37C on a 250 rpm shaker for 6-8 hours
-
* Length of time on ice prior to transformation may make a big difference
+
# At around 6 PM, pour the water out of your (3) 1L flasks and add 250mL of SOB to each.  Label them 10, 4 and 2.
-
* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference.  Let's see.
+
# Innoculate these cultures with 10, 4, and 2 mL of the starter culture, respectively.
-
* Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
+
# Incubate at room temperature (the lower the better, down to around 20C) at 100 rpm
-
* My lab uses LB for instead of SOB media...it seems to work fine for them--[[User:Melissali|mel]] 18:10, 14 June 2007 (EDT)
+
# Put 140 .5mL microcentrifuge tubes in a rack, and place them in a sealed bag in the -20 or -40 freezer overnight.
 +
# Label two pipet tip boxes or similar with the strain, your initials, the date, and either 150 uL or 100 uL.  Store at -40C overnight
 +
 
 +
===Day 3===
 +
# Thaw 100 mL of Inoue transformation buffer.  Keep on ice.
 +
# Prechill the centrifuge to 4 degrees
 +
# Take samples of each of the (3) overnight cultures, and return to the shaker.  Take the OD600 of each on a spectrometer.
 +
# If one of the cultures is at .55 or just over, proceed.  We had a good batch from a .575 OD Otherwise, keep taking ODs every 45 minutes until one of the cultures reaches .55
 +
# Place the .55 OD600 culture on ice for 10 minutes.  Keep the culture(s) that are below .55 on the shaker in case you mess up.
 +
# Pour the chilled .55 culture into a sterile 250 mL centrifuge bottle
 +
# Centrifuge at 2500''g'' for 10 minutes at 4C.  Balance with another bottle with 250mL water.  The pellet may have some black streaks in it- this is normal.
 +
# Drain the medium and aspirate any remaining medium out of the bottle if you have a vacuum trap. Otherwise just keep the bottle upside down on paper towels for 2 minutes.
 +
# Add 80mL ice-cold Inoue transformation bufferm, and swirl to resuspend.
 +
# Spin down as above, remove buffer as above.
 +
# Add 20 ml of cold ice-cold Inoue buffer, swirl to resuspend.
 +
# Add 1.5mL of high-purity DMSO (99.9%- not quite electrophoresis grade-worked good), swirl to mix. 
 +
# Incubate on ice for 10 minutes.  In the meantime, get a few inches of liquid nitrogen in an ice bucket
 +
# Put your racks of microcentrifuge tubes on ice in an autoclave tray
 +
# With a genomic (wide-mouth) tip, allocate 150uL of cells into 2/3 of the tubes, and 100uL into the other third.  Or allocate 50 uL into each, but it will take a while.  You're always going to be doing 2 or 3 50 uL transformations at a time anyway, with + and/or - controls  Get someone to help you freeze as you allocate.
 +
# To freeze, submerge the tip of the tubes in the liquid nitrogen for a few seconds, then let go so the tubes are floating in the bath.  Wear cryo gloves and be careful- this stuff can freeze your skin off mighty quick.
 +
# scoop the tubes out of the bath with a pipette tip box lid, and pour them into the cold labeled tip boxes from the freezer.
 +
# Immediately place the boxes of competent cells at -80C
-
==Related topics & references==
 
-
*[[Preparing chemically competent cells]]
 
-
*[[TSS|Preparing TSS buffer]]
 
-
*[[Transforming chemically competent cells]]''
 
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*[[Electrocompetent cells|Preparing electrocompetent cells]]
 
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*[[Electroporation]]
 
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*[[TB buffer]]
 
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*[[Transforming chemically competent cells (Inoue)]]
 
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*[[Bacterial cell culture]]
 
-
Original protocol from Inoue et al. <cite> Inoue90 </cite>.  Useful comments and speculation  about reducing agents in <cite>Hengen96</cite>.
+
Original protocol from Inoue et al.  
<biblio>
<biblio>

Latest revision as of 08:51, 28 October 2008



Contents

Making Ultracompetent Cells

  • Transformation can be a major roadblock in assembling DNA constructs. Ligations can be hard to transform, especially with large inserts. DNA blotted on paper from the registry tends to be even harder. We have had a great experience with the following protocol from Inoue, getting near lawns of transformants from ligations:
  • Based on the OpenWetWare page: http://openwetware.org/wiki/Preparing_chemically_competent_cells_(Inoue)
  • Makes about 25mL, or enough for nearly 500 50 uL transformations

Materials

  • Plate of cells streaked for single colonies. We used DH5alpha.
  • [http://openwetware.org/wiki/SOB SOB]
  • Ice
  • [http://openwetware.org/wiki/TB_buffer TB buffer]
  • DMSO (99.9%)
  • Liquid nitrogen

Glassware & equipment

  • (3)1 liter Erlenmeyer flasks (no detergent residue- we rinsed well with pure water, then autoclaved 2/3 full of pure water, emptying just before use
  • (1) 250 mL Erlenmeyer flask
  • 250 mL centrifuge bottles, autoclaved
  • Refrigerated centrifuge

Preparation

Day 1

  1. Pour LB plates (no antibiotic)
  2. In the afternoon, streak a plate of DH5alpha from frozen stock. (We usually just use a sterile 200 ul pipette tip, scrape some frozen cells off the top, melt them in a pool near an edge of the plate, return the still-frozen stock to the -80C freezer, and streak back and forth across the surface.)
  3. Incubate plate inverted at 37C overnight (14-20 hours)
  4. Make 1L of SOB medium in a bottle without detergent residue
  5. Rinse out the clean flasks very well to remove detergent residue- (3) 1L, (1) 250 mL. Fill each 2/3 with pure water and autoclave. Keep the water in overnight.

Day 2

  1. At around 11 AM, pour the water out of the 250 mL flask and add 25mL SOB
  2. Innoculate with one colony from the LB plate
  3. Grow this starter culture at 37C on a 250 rpm shaker for 6-8 hours
  4. At around 6 PM, pour the water out of your (3) 1L flasks and add 250mL of SOB to each. Label them 10, 4 and 2.
  5. Innoculate these cultures with 10, 4, and 2 mL of the starter culture, respectively.
  6. Incubate at room temperature (the lower the better, down to around 20C) at 100 rpm
  7. Put 140 .5mL microcentrifuge tubes in a rack, and place them in a sealed bag in the -20 or -40 freezer overnight.
  8. Label two pipet tip boxes or similar with the strain, your initials, the date, and either 150 uL or 100 uL. Store at -40C overnight

Day 3

  1. Thaw 100 mL of Inoue transformation buffer. Keep on ice.
  2. Prechill the centrifuge to 4 degrees
  3. Take samples of each of the (3) overnight cultures, and return to the shaker. Take the OD600 of each on a spectrometer.
  4. If one of the cultures is at .55 or just over, proceed. We had a good batch from a .575 OD Otherwise, keep taking ODs every 45 minutes until one of the cultures reaches .55
  5. Place the .55 OD600 culture on ice for 10 minutes. Keep the culture(s) that are below .55 on the shaker in case you mess up.
  6. Pour the chilled .55 culture into a sterile 250 mL centrifuge bottle
  7. Centrifuge at 2500g for 10 minutes at 4C. Balance with another bottle with 250mL water. The pellet may have some black streaks in it- this is normal.
  8. Drain the medium and aspirate any remaining medium out of the bottle if you have a vacuum trap. Otherwise just keep the bottle upside down on paper towels for 2 minutes.
  9. Add 80mL ice-cold Inoue transformation bufferm, and swirl to resuspend.
  10. Spin down as above, remove buffer as above.
  11. Add 20 ml of cold ice-cold Inoue buffer, swirl to resuspend.
  12. Add 1.5mL of high-purity DMSO (99.9%- not quite electrophoresis grade-worked good), swirl to mix.
  13. Incubate on ice for 10 minutes. In the meantime, get a few inches of liquid nitrogen in an ice bucket
  14. Put your racks of microcentrifuge tubes on ice in an autoclave tray
  15. With a genomic (wide-mouth) tip, allocate 150uL of cells into 2/3 of the tubes, and 100uL into the other third. Or allocate 50 uL into each, but it will take a while. You're always going to be doing 2 or 3 50 uL transformations at a time anyway, with + and/or - controls Get someone to help you freeze as you allocate.
  16. To freeze, submerge the tip of the tubes in the liquid nitrogen for a few seconds, then let go so the tubes are floating in the bath. Wear cryo gloves and be careful- this stuff can freeze your skin off mighty quick.
  17. scoop the tubes out of the bath with a pipette tip box lid, and pour them into the cold labeled tip boxes from the freezer.
  18. Immediately place the boxes of competent cells at -80C


Original protocol from Inoue et al.

<biblio>

  1. Inoue90 pmid=2265755
  2. Hengen96 pmid=8851666

</biblio>

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