Alm:Protoplasting - Revision history

ClarkeS: /* Materials for 1 protoplasting run */

ClarkeS — Wed, 04 Apr 2007 15:09:58 GMT

Materials for 1 protoplasting run

←Older revision		Revision as of 15:09, 4 April 2007
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	==Materials for 1 protoplasting run==		==Materials for 1 protoplasting run==

-	*Tris-HCl buffer, pH 8.0, washing ~~and resuspending~~, ~~~90 ml~~	+	*W1: Tris-HCl buffer, pH 8.0, 0.01M, washing solution 1, 3x wash volume so ~30ml
-	*EDTA, 0.1M, 3.~~3 ml~~	+	*R1: Tris-HCl + 0.5M sucrose, 30ml
-	*sucrose, 0.5M	+	*EDTA, 0.1M, 3.3ml
-	*SMM, ~~divided~~	+	*SMM = sucrose, 0.5M , , 30 + 3x wash volume, so ~60ml
-	**SMM + lysozyme 1 mg/ml	+	**W2: SMM for washing, 3x wash volume so ~30ml
-	*TSB + sucrose	+	**R2: SMM + lysozyme 1 mg/ml, 30ml
		+
		+	*R3: TSB + 0.5M sucrose, resuspension and dilution for plating, also need some for plating control with original cells 50 ml?
		+	*TSB + 0.5M sucrose + 0.8% agarose

	*50 ml Falcon tube, 1 per protoplast type		*50 ml Falcon tube, 1 per protoplast type

ClarkeS: /* Materials for 1 round */

ClarkeS — Tue, 03 Apr 2007 18:47:42 GMT

Materials for 1 round

←Older revision		Revision as of 18:47, 3 April 2007
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-	==Materials for 1 ~~round~~==	+	==Materials for 1 protoplasting run==

		+	*Tris-HCl buffer, pH 8.0, washing and resuspending, ~90 ml
		+	*EDTA, 0.1M, 3.3 ml
	*sucrose, 0.5M		*sucrose, 0.5M
	*SMM, divided		*SMM, divided
-	**SMM + lysozyme	+	**SMM + lysozyme 1 mg/ml
		+	*TSB + sucrose
		+
		+	*50 ml Falcon tube, 1 per protoplast type
		+	*25 ml serological pipets
		+	*P1000
		+	*P20
		+	*plates

	===Regeneration:===		===Regeneration:===

-	soft agar (0.8%) plates containing	+	soft agar (0.8%) plates containing

	In some cases, protoplasts were resus-		In some cases, protoplasts were resus-
Line 21:		Line 30:
	nutritional requirements of the auxotrophic strains used,		nutritional requirements of the auxotrophic strains used,
	and then poured onto soft agar plates of the same		and then poured onto soft agar plates of the same
-	composition.	+	composition.
-		+

	==Overview==		==Overview==

ClarkeS: /* Permeabilize cell wall */

ClarkeS — Mon, 02 Apr 2007 20:22:03 GMT

Permeabilize cell wall

←Older revision		Revision as of 20:22, 2 April 2007
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	===Permeabilize cell wall===		===Permeabilize cell wall===
-	#~~Step 1~~	+	#Pellet, rinse with SMM, resuspend in SMM + lysozyme
-	#~~Step 2~~	+	#Shake and incubate
-	#*~~Step 2 has some additional information that goes with it. i.e. Keep at 4°C.~~	+	*No current step to stop lysozyme
-	~~#Step 3~~	+	*Should have protoplasts now
-	~~##Step 3 has multiple sub-steps within it.~~	+
-	~~##Enumerate each of those.~~	+

	===Optional: Fuse protoplasts===		===Optional: Fuse protoplasts===

ClarkeS: /* Strip outer membrane */

ClarkeS — Mon, 02 Apr 2007 20:19:17 GMT

Strip outer membrane

←Older revision		Revision as of 20:19, 2 April 2007
Line 41:		Line 41:
	==Procedure==		==Procedure==
	===Strip outer membrane===		===Strip outer membrane===
-	#~~Step 1~~	+	#Pellet cells
-	#~~Step 2~~	+	#Pour off supernatant
-	#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.	+	#Resuspend in Tris-HCl
-	#~~Step 3~~	+	#Add EDTA slowly
-	#~~#Step 3 has multiple sub-steps within it.~~	+	#Shake
-	#~~#Enumerate each of those.~~	+	#Pellet and rinse

	===Permeabilize cell wall===		===Permeabilize cell wall===

ClarkeS: /* Procedure */ outlined

ClarkeS — Mon, 02 Apr 2007 20:04:35 GMT

Procedure: outlined

←Older revision		Revision as of 20:04, 2 April 2007
Line 40:		Line 40:

	==Procedure==		==Procedure==
		+	===Strip outer membrane===
		+	#Step 1
		+	#Step 2
		+	#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
		+	#Step 3
		+	##Step 3 has multiple sub-steps within it.
		+	##Enumerate each of those.
		+
		+	===Permeabilize cell wall===
		+	#Step 1
		+	#Step 2
		+	#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
		+	#Step 3
		+	##Step 3 has multiple sub-steps within it.
		+	##Enumerate each of those.
		+
		+	===Optional: Fuse protoplasts===
		+	#Mix aliquots of protoplasts of each parent strain
		+	#Add PEG
		+	#Step 3
		+
		+	===Regenerate protoplasts===
	#Step 1		#Step 1
	#Step 2		#Step 2

ClarkeS: /* Contact */

ClarkeS — Mon, 02 Apr 2007 19:33:33 GMT

Contact

←Older revision		Revision as of 19:33, 2 April 2007
Line 65:		Line 65:

	==Contact==		==Contact==
-	*~~Who has experience with this protocol?~~	+	*[[Sean Clarke]]

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

ClarkeS: roughed creation of page

ClarkeS — Mon, 02 Apr 2007 19:30:29 GMT

roughed creation of page

New page

==Materials for 1 round==

*sucrose, 0.5M
*SMM, divided
**SMM + lysozyme

===Regeneration:===

soft agar (0.8%) plates containing

In some cases, protoplasts were resus-
pended in LB medium containing 0.5 M sucrose and
spread onto soft agar plates containing LB medium and
0.5 M sucrose with autoclaved plastic spreaders. The
plates were subsequently incubated at either 25 or 37 1C.

In other cases, the protoplasts were diluted into molten
soft agar at 45 1C containing either LB medium and
0.5 M sucrose or M9 medium, 0.5 M sucrose, and either
Leu (100 mg/ml) or Arg (100 mg/ml), depending on the
nutritional requirements of the auxotrophic strains used,
and then poured onto soft agar plates of the same
composition.

==Overview==

Replace this sentence with a brief description of the protocol and its goal.

==Materials==
List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

*supply 1 (i.e. tubes of a certain size? spreaders?)
*reagent 1
*X μL reagent 2
**component A (reagent 2 is made up of multiple components)
**component B
*equipment 1
*equipment 2

==Procedure==
#Step 1
#Step 2
#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
#Step 3
##Step 3 has multiple sub-steps within it.
##Enumerate each of those.

==Notes==
#List troubleshooting tips here.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

==References==
'''Relevant papers and books'''
<biblio>

</biblio>

==Contact==
*Who has experience with this protocol?

or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].


[[Category:Protocol]]