TOP10 chemically competent cells - Revision history

Tk at 23:05, 17 February 2013

2013-02-17T23:05:14Z

←Older revision		Revision as of 23:05, 17 February 2013
Line 20:		Line 20:
	* sterile filter and store at 4°C		* sterile filter and store at 4°C
	* slight dark precipitate appears not to affect its function		* slight dark precipitate appears not to affect its function
		+	* Note: you can buy pre-made CCMB80 buffer from Teknova

	==Procedure==		==Procedure==

Tk at 22:23, 17 February 2013

2013-02-17T22:23:44Z

←Older revision		Revision as of 22:23, 17 February 2013
Line 44:		Line 44:

	===Preparing competent cells===		===Preparing competent cells===
-	* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.5	+	* 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.		** This takes approximately 16 hours.
	** Controlling the temperature makes this a more reproducible process, but is not essential.		** 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		** 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		** Aim for lower, not higher OD if you can't hit this mark
-	* Centrifuge at ~~5000rpm~~ at 4°C for 10 minutes in a flat bottom centrifuge bottle.	+	* Centrifuge at 3000rpm at 4°C for 10 minutes in a flat bottom centrifuge bottle.
	** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend		** 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		** It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer
-	* Discard supernatant by pouring out slowly and pipeting ~~remains~~	+	* Discard supernatant by pouring out slowly and pipeting remaining supernatent
	* Gently resuspend in 80 ml of ice cold CCMB80 buffer		* Gently resuspend in 80 ml of ice cold CCMB80 buffer
	** sometimes this is less than completely gentle. It still works.		** sometimes this is less than completely gentle. It still works.

Cong T. Trinh: /* Preparing competent cells */

2011-08-26T02:51:54Z

Preparing competent cells

Cong T. Trinh: /* Preparing competent cells */

2011-08-25T01:07:18Z

Preparing competent cells

←Older revision		Revision as of 01:07, 25 August 2011
Line 44:		Line 44:

	===Preparing competent cells===		===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	+	* Inoculate 10 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.5
	** This takes approximately 16 hours.		** This takes approximately 16 hours.
	** Controlling the temperature makes this a more reproducible process, but is not essential.		** 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		** 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		** 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.	+	* Centrifuge at 4000rpm at 4°C for 10 minutes in a flat bottom centrifuge bottle.
	** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend		** 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		** 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	+	* Discard supernatant by pouring out slowly and pipeting remaining out
		+	* Gently resuspend in 10 ml of ice cold CCMB80 buffer
	** sometimes this is less than completely gentle. It still works.		** sometimes this is less than completely gentle. It still works.
	* Incubate on ice 20 minutes		* Incubate on ice 20 minutes
-	* Centrifuge again at 4°C and ~~resuspend~~ in 10 ml of ice cold CCMB80 buffer.	+	* Centrifuge again at 4°C and discard supernatant as described above.
		+	* Resuspend in 10 ml of ice cold CCMB80 buffer.
	* Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.		* Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
-	* Add chilled CCMB80 to yield a final OD of 1.0~~-1.5~~ in this test.	+	* Add chilled CCMB80 to yield a final OD of ~3.0 in this test.
-	* Incubate on ice for 20 minutes	+
	* Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates		* Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
	* Store at -80°C indefinitely.		* Store at -80°C indefinitely.

Torsten Waldminghaus at 17:10, 23 February 2009

2009-02-23T17:10:10Z

←Older revision		Revision as of 17:10, 23 February 2009
Line 1:		Line 1:
		+	{{back to protocols}}
	==Overview==		==Overview==
	This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [[Media:pat6855494.pdf \| Bloom05 patent]] as well. This protocol has been tested on TOP10, MachI and [[Talk:TOP10 chemically competent cells\|BL21(DE3)]] cells. See [[Bacterial Transformation]] for a more general discussion of other techniques. The [[Media:pat6960464.pdf \| Jesse '464 patent]] describes using this buffer for DH5α cells. The [[Media:pat6709852.pdf \| Bloom04]] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.		This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [[Media:pat6855494.pdf \| Bloom05 patent]] as well. This protocol has been tested on TOP10, MachI and [[Talk:TOP10 chemically competent cells\|BL21(DE3)]] cells. See [[Bacterial Transformation]] for a more general discussion of other techniques. The [[Media:pat6960464.pdf \| Jesse '464 patent]] describes using this buffer for DH5α cells. The [[Media:pat6709852.pdf \| Bloom04]] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

Felix Moser: /* Measurement of competence */

2008-07-07T15:15:26Z

Measurement of competence

←Older revision		Revision as of 15:15, 7 July 2008
Line 77:		Line 77:
	* Plate 20 μl on AMP plates using sterile 3.5 mm glass beads		* Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
	** Good cells should yield around 100 - 400 colonies		** Good cells should yield around 100 - 400 colonies
-	** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup> ~~cfu~~/µgDNA	+	** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup>/µgDNA
	**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup> cfu/µgDNA		**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup> cfu/µgDNA

Felix Moser: /* Measurement of competence */

2008-07-07T15:12:38Z

Measurement of competence

←Older revision		Revision as of 15:12, 7 July 2008
Line 78:		Line 78:
	** Good cells should yield around 100 - 400 colonies		** Good cells should yield around 100 - 400 colonies
	** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup> cfu/µgDNA		** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup> cfu/µgDNA
-	**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup>	+	**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup> cfu/µgDNA

	==5x Ligation Adjustment Buffer==		==5x Ligation Adjustment Buffer==

Felix Moser: /* Measurement of competence */

2008-07-07T15:11:40Z

Measurement of competence

←Older revision		Revision as of 15:11, 7 July 2008
Line 76:		Line 76:
	** Ampicillin and kanamycin appear to do fine with 1 hour growth		** Ampicillin and kanamycin appear to do fine with 1 hour growth
	* Plate 20 μl on AMP plates using sterile 3.5 mm glass beads		* Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
-	* Transformation efficiency is 15 x colony count x 10<sup>5</sup>	+	** Good cells should yield around 100 - 400 colonies
		+	** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup> cfu/µgDNA
	**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup>		**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup>

Felix Moser: /* Preparing competent cells */

2008-07-07T15:10:36Z

Preparing competent cells

←Older revision		Revision as of 15:10, 7 July 2008
Line 62:		Line 62:
	** Flash freezing does not appear to be necessary		** Flash freezing does not appear to be necessary
	* Test competence (see below)		* Test competence (see below)
-	** Good cells should yield around 100 - 400 colonies
	* 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.		* 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.

Meaganl at 06:21, 21 June 2008

2008-06-21T06:21:36Z

←Older revision		Revision as of 06:21, 21 June 2008
Line 78:		Line 78:
	* Plate 20 μl on AMP plates using sterile 3.5 mm glass beads		* Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
	* Transformation efficiency is 15 x colony count x 10<sup>5</sup>		* Transformation efficiency is 15 x colony count x 10<sup>5</sup>
		+	**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup>

	==5x Ligation Adjustment Buffer==		==5x Ligation Adjustment Buffer==

←Older revision		Revision as of 02:51, 26 August 2011
Line 44:		Line 44:

	===Preparing competent cells===		===Preparing competent cells===
-	* Inoculate 10 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.5	+	* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.5
	** This takes approximately 16 hours.		** This takes approximately 16 hours.
	** Controlling the temperature makes this a more reproducible process, but is not essential.		** 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		** 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		** Aim for lower, not higher OD if you can't hit this mark
-	* Centrifuge at ~~4000rpm~~ at 4°C for 10 minutes in a flat bottom centrifuge bottle.	+	* Centrifuge at 5000rpm at 4°C for 10 minutes in a flat bottom centrifuge bottle.
	** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend		** 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		** It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer
-	* Discard supernatant by pouring out slowly and pipeting ~~remaining out~~	+	* Discard supernatant by pouring out slowly and pipeting remains
-	* Gently resuspend in 10 ml of ice cold CCMB80 buffer	+	* Gently resuspend in 80 ml of ice cold CCMB80 buffer
	** sometimes this is less than completely gentle. It still works.		** sometimes this is less than completely gentle. It still works.
	* Incubate on ice 20 minutes		* Incubate on ice 20 minutes
Line 59:		Line 59:
	* Resuspend in 10 ml of ice cold CCMB80 buffer.		* Resuspend in 10 ml of ice cold CCMB80 buffer.
	* Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.		* Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
-	* Add chilled CCMB80 to yield a final OD of ~3.0 in this test.	+	* Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
	* Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates		* Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
	* Store at -80°C indefinitely.		* Store at -80°C indefinitely.
	** Flash freezing does not appear to be necessary		** Flash freezing does not appear to be necessary
	* Test competence (see below)		* 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.	+	* 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===		===Measurement of competence===