Questions and Answers - Revision history

Rachel Davis at 13:18, 1 April 2009

Rachel Davis — Wed, 01 Apr 2009 13:18:27 GMT

←Older revision		Revision as of 13:18, 1 April 2009
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	''Post new questions at the top of the list''		''Post new questions at the top of the list''
	{{Discussion}}		{{Discussion}}
		+
		+	==Condensing linear DNA==
		+	*'''[[User:Rachel Davis\|Rachel Davis]] 09:18, 1 April 2009 (EDT)''':I would like to know if there is any decent assay to condense linear DNA in vitro?
		+	I read some studies about condensin from Kimura, K. and Hirano, T. but they worked with circular DNA from bacteria. I also heard something about polymers, which induced condensation of DNA supercoils.

	==Transduction question==		==Transduction question==

Rachel Davis at 14:03, 31 March 2009

Rachel Davis — Tue, 31 Mar 2009 14:03:39 GMT

←Older revision		Revision as of 14:03, 31 March 2009
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	==Reply==		==Reply==
	*'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 04:14, 31 March 2009 (EDT)''': I think that the number of colonies is the only measure of transduction success, for sure from the list of suggestions you wrote.		*'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 04:14, 31 March 2009 (EDT)''': I think that the number of colonies is the only measure of transduction success, for sure from the list of suggestions you wrote.
		+
		+	*'''[[User:Rachel Davis\|Rachel Davis]] 10:03, 31 March 2009 (EDT)''': Thanks so much.

	==E.coli MC4100 thi- genotype==		==E.coli MC4100 thi- genotype==

Torsten Waldminghaus: /* Transduction question */

Torsten Waldminghaus — Tue, 31 Mar 2009 08:14:37 GMT

Transduction question

←Older revision		Revision as of 08:14, 31 March 2009
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	==Transduction question==		==Transduction question==
	*'''[[User:Rachel Davis\|Rachel Davis]] 21:28, 30 March 2009 (EDT)''':To measure the success of transduction, what, other than the number of colonies, are plausible dependent variables? Size of the colonies? Distance between them? And why would such variables they imply success of transduction? Thanks.		*'''[[User:Rachel Davis\|Rachel Davis]] 21:28, 30 March 2009 (EDT)''':To measure the success of transduction, what, other than the number of colonies, are plausible dependent variables? Size of the colonies? Distance between them? And why would such variables they imply success of transduction? Thanks.
		+
		+	==Reply==
		+	*'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 04:14, 31 March 2009 (EDT)''': I think that the number of colonies is the only measure of transduction success, for sure from the list of suggestions you wrote.
		+
	==E.coli MC4100 thi- genotype==		==E.coli MC4100 thi- genotype==
	I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.		I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.

Rachel Davis at 01:28, 31 March 2009

Rachel Davis — Tue, 31 Mar 2009 01:28:59 GMT

←Older revision		Revision as of 01:28, 31 March 2009
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	''Post new questions at the top of the list''		''Post new questions at the top of the list''
	{{Discussion}}		{{Discussion}}
		+
		+	==Transduction question==
		+	*'''[[User:Rachel Davis\|Rachel Davis]] 21:28, 30 March 2009 (EDT)''':To measure the success of transduction, what, other than the number of colonies, are plausible dependent variables? Size of the colonies? Distance between them? And why would such variables they imply success of transduction? Thanks.
	==E.coli MC4100 thi- genotype==		==E.coli MC4100 thi- genotype==
	I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.		I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.

Torsten Waldminghaus at 11:00, 24 February 2009

Torsten Waldminghaus — Tue, 24 Feb 2009 11:00:35 GMT

←Older revision		Revision as of 11:00, 24 February 2009
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	I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.		I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.
	I've had a look at the genotype on this site, the ATCC, and EcoliWiki, all I've gotten is thi-, a literature search turned up even less. Does anyone know what gene has been mutated/deleted in the thi- MC4100 genotype?		I've had a look at the genotype on this site, the ATCC, and EcoliWiki, all I've gotten is thi-, a literature search turned up even less. Does anyone know what gene has been mutated/deleted in the thi- MC4100 genotype?
		+
		+	===Reply===
		+	*'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 06:00, 24 February 2009 (EST)''':I'm pretty sure that it is the thiE gene (synonyme thiA). This codes for the last step in the thiamine biosythesis and if you delete a gene coding for some earlier enzymes you would need to give E. coli more than thiamine. By the way, if you want to clone the pspA promotor you might find one of my papers interesting where I actually made some psp fusions:
		+	<biblio>
		+	#waldminghaus-2007 pmid=17647020
		+	</biblio>
		+	I would be happy to send you any of the mentioned plasmids if they could help you.

	==About Trizol==		==About Trizol==

Julian Spagnuolo at 03:57, 24 February 2009

Julian Spagnuolo — Tue, 24 Feb 2009 03:57:45 GMT

←Older revision		Revision as of 03:57, 24 February 2009
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	''Post new questions at the top of the list''		''Post new questions at the top of the list''
	{{Discussion}}		{{Discussion}}
		+	==E.coli MC4100 thi- genotype==
		+	I'm trying to create a pspA reporter plasmid by linking the psp promoter using the thi- genotype of E.coli MC4100 as an auxotrophic marker, however I'm having 'a little' trouble finding out which gene has actually been deleted or mutated so that I can incorporate it into my plasmid.
		+	I've had a look at the genotype on this site, the ATCC, and EcoliWiki, all I've gotten is thi-, a literature search turned up even less. Does anyone know what gene has been mutated/deleted in the thi- MC4100 genotype?
		+
	==About Trizol==		==About Trizol==
	I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!		I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!

Torsten Waldminghaus at 21:26, 18 February 2009

Torsten Waldminghaus — Wed, 18 Feb 2009 21:26:34 GMT

←Older revision		Revision as of 21:26, 18 February 2009
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	I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!		I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!

-	====	+	==Lambda red recombination==
	Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?		Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?
		+
		+	===Reply===
		+	*'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 16:26, 18 February 2009 (EST)''': You can find a nice description of the primer design in the OpenWetWare protocol [[Recombineering/Lambda red-mediated gene replacement]]. The part of the primer that is complementary to the resistance gene is relevant for your PCR parameters. The part of the primer that is complementary to the gene you want to desrupt only hangs arround as a tail in the PCR reaction. If you think about Tm as basis for PCR you should only consider the bp complementary to the resistence gene.

	==Automatic colony counter==		==Automatic colony counter==

Leo Tang: /* About Trizol */

Leo Tang — Tue, 20 Jan 2009 00:03:44 GMT

About Trizol

←Older revision		Revision as of 00:03, 20 January 2009
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	I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!		I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!

-		+	====
	Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?		Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?

Leo Tang at 00:03, 20 January 2009

Leo Tang — Tue, 20 Jan 2009 00:03:19 GMT

←Older revision		Revision as of 00:03, 20 January 2009
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	''Post new questions at the top of the list''		''Post new questions at the top of the list''
	{{Discussion}}		{{Discussion}}
		+	==About Trizol==
		+	I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[http://openwetware.org/wiki/RNA_extraction_using_trizol/tri]]). In protocols Guanidinium Thioyanate(W.M. 118.2) final concentration is 0.8M, but 118.2g is used in 1L Trizol. Ammonium thiocyanate (W.M. 76.1) is 76.1g in Trizol, however, they said the final concentration is 0.4M. I can not understant that. Thank you for your answer!
		+
		+
	Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?		Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?

Ying Qi: the primer of red recombinase

Ying Qi — Tue, 25 Mar 2008 00:19:56 GMT

the primer of red recombinase

←Older revision		Revision as of 00:19, 25 March 2008
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	''Post new questions at the top of the list''		''Post new questions at the top of the list''
	{{Discussion}}		{{Discussion}}
		+	Does anybody have experience in red recombinase? We are recently get started and have little experience in primer design. When add the homology sequence, the primer is long, what should we do if the primer have a high Tm, or too much hairpin and Dimer? How do set the PCR parameters?

	==Automatic colony counter==		==Automatic colony counter==