McClean:Competent Cells - Revision history

Ping Xu at 20:04, 23 April 2013

Ping Xu — Tue, 23 Apr 2013 20:04:39 GMT

←Older revision		Revision as of 20:04, 23 April 2013
Line 19:		Line 19:

	==Preparation==		==Preparation==
-	# Pick a single colonies (2-3 mm dia) from your source plate and inoculate 25 ml of sterile LB medium (do not use LB) in a 250mL flask. Incubate the culture in 37°C for 6-8 hours with vigorous shaking at 250rpm.	+	# Pick a single colonies (2-3 mm dia) from your source plate and inoculate 25 ml of sterile LB medium (do not use SOB) in a 250mL flask. Incubate the culture in 37°C for 6-8 hours with vigorous shaking at 250rpm.
	# After the culture is incubated 6-8 hours, before you leave the lab,use this culture to inoculate three 1-liter flasks, each containing 250ml of SOB. The first flask receives 10ml of starter culture, the second receives 4ml and the third receives 2ml.Incubate all three flasks at room temperature overnight with moderate shaking at 200rpm.		# After the culture is incubated 6-8 hours, before you leave the lab,use this culture to inoculate three 1-liter flasks, each containing 250ml of SOB. The first flask receives 10ml of starter culture, the second receives 4ml and the third receives 2ml.Incubate all three flasks at room temperature overnight with moderate shaking at 200rpm.
	# In the next morning, check OD600 with all three flasks.Pick the flask OD600 closest to 0.55. If the flask is a little over 0.55, put the flask in ice-water bath right away; otherwise keep monitor OD600 till it reaches 0.55.		# In the next morning, check OD600 with all three flasks.Pick the flask OD600 closest to 0.55. If the flask is a little over 0.55, put the flask in ice-water bath right away; otherwise keep monitor OD600 till it reaches 0.55.

Ping Xu at 20:24, 31 October 2011

Ping Xu — Mon, 31 Oct 2011 20:24:01 GMT

←Older revision		Revision as of 20:24, 31 October 2011
Line 4:		Line 4:
	==Materials==		==Materials==
	* Plate of cells streaked for single colonies		* Plate of cells streaked for single colonies
		+	* LB
	* [[SOB]]		* [[SOB]]
	* Ice		* Ice
	* [[TB buffer]]		* [[TB buffer]]
	* DMSO		* DMSO
-	* ~~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~~)	+	* 250 ml flask (no detergent residue, autoclaved)
-	* ~~220~~ ml ~~conical centrifuge tubes~~ BD ~~35 2075~~	+	* 1 liter flask (no detergent residue, autoclaved)
-	* Eppendorf ~~5410R~~ refrigerated centrifuge with conical adapters	+	* 50 ml BD Falcon tubes
		+	* Eppendorf 5810R refrigerated centrifuge with conical adapters

	==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~~.	+	# Pick a single colonies (2-3 mm dia) from your source plate and inoculate 25 ml of sterile LB medium (do not use LB) in a 250mL flask. Incubate the culture in 37°C for 6-8 hours with vigorous shaking at 250rpm.
-	# ~~Grow~~ to ~~an OD~~ of 0.6 with ~~vigorous~~ shaking ~~(200-250 rpm)~~ at ~~18 degrees (important)~~. ~~This~~ is ~~slow~~ -- ~~approximately 35-40 hours~~.	+	# After the culture is incubated 6-8 hours, before you leave the lab,use this culture to inoculate three 1-liter flasks, each containing 250ml of SOB. The first flask receives 10ml of starter culture, the second receives 4ml and the third receives 2ml.Incubate all three flasks at room temperature overnight with moderate shaking at 200rpm.
		+	# In the next morning, check OD600 with all three flasks.Pick the flask OD600 closest to 0.55. If the flask is a little over 0.55, put the flask in ice-water bath right away; otherwise keep monitor OD600 till it reaches 0.55.
		+	# Incubate the culture in ice-water bath for 10 minutes.
	# Prechill the centrifuge to 4 degrees		# Prechill the centrifuge to 4 degrees
-	# ~~Remove from~~ the ~~incubator and place on ice for 10 minutes~~	+	# Split the culture to five 50ml tubes.
-	~~# Transfer~~ to ~~two 220 ml centrifuge~~ tubes ~~and spin~~ at ~~3220 x g~~ for 10 minutes ~~at 4 degrees~~	+	# Centrifuge at 3000g for 10 minutes.
-	# 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~~.	+	# Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
-	# ~~Place on ice~~ for 10 minutes	+	# Resuspend each pellet in 40 ml of ice cold TB. Resuspend the cells by swirling rather than pipetting or vortexing.
-	# ~~Spin down as above~~.	+	# Centrifuge at 3000g for 10 minutes.
-	# ~~While spinning, add 1.~~4 ml of ~~DMSO~~ to 18.~~6 ml of TB (7% DMSO mixture)~~	+	# Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
-	# ~~Resuspend each pellet in 20~~ ml of ~~cold TB-~~DMSO ~~mixture~~	+	# Add 4 ml ice-cold TB buffer to each tube, resuspend the cell pellets then combine all of them to one tube and put the tube on ice.
		+	# Add 1.5 ml of DMSO to the tube and mix.
	# Incubate on ice for 10 minutes		# Incubate on ice for 10 minutes
-	# Dispense cells into ~~pre-chilled~~ tubes	+	# Dispense cells into tubes and drop the tubes to liquid nitrogen.
-	# ~~Freeze cells in a dry ice / ethanol bath and store~~ at -80 degrees ~~indefinitely~~	+	# Store at -80 degrees.

	==Thoughts on improvements==		==Thoughts on improvements==
Line 38:		Line 43:
	* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.		* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.
	* Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.		* Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
-	* 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)

	==Related topics & references==		==Related topics & references==

Ping Xu: New page: {{back to protocols}} This protocol is used for preparing competent cells for transformation. ==Materials== * Plate of cells streaked for single colonies * SOB * Ice * TB buffer *...

Ping Xu — Mon, 31 Oct 2011 19:17:33 GMT

New page: {{back to protocols}} This protocol is used for preparing competent cells for transformation. ==Materials== * Plate of cells streaked for single colonies * SOB * Ice * TB buffer *...

New page

{{back to protocols}}
This protocol is used for preparing competent cells for transformation.

==Materials==
* Plate of cells streaked for single colonies
* [[SOB]]
* Ice
* [[TB buffer]]
* DMSO
* Dry Ice (or liquid nitrogen)
*

==Glassware & equipment==
* 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water)
* 220 ml conical centrifuge tubes BD 35 2075
* Eppendorf 5410R refrigerated centrifuge with conical adapters

==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.
# Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours.
# Prechill the centrifuge to 4 degrees
# Remove from the incubator and place on ice for 10 minutes
# Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
# 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.
# 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==
* "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
* They also control pH at 7.5, which may be a major issue
* Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
* Length of time on ice prior to transformation may make a big difference
* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.
* Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
* 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)

==Related topics & references==
*[[Preparing chemically competent cells]]
*[[TSS|Preparing TSS buffer]]
*[[Transforming chemically competent cells]]''
*[[Electrocompetent cells|Preparing electrocompetent cells]]
*[[Electroporation]]
*[[TB buffer]]
*[[Transforming chemically competent cells (Inoue)]]
*[[Bacterial cell culture]]

Original protocol from Inoue et al. <cite> Inoue90 </cite>. Useful comments and speculation about reducing agents in <cite>Hengen96</cite>.

<biblio>
#Inoue90 pmid=2265755
#Hengen96 pmid=8851666
</biblio>

[[Category:Protocol]]
[[Category:Escherichia coli]]