Questions and Answers

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Thought this might be a good thing to add as a general forum for questions people may have. Wiki folk could check it out periodically and offer help if they can. Answers could be posted here, or refer to appropriate sections of the wiki. Additionally, it could put people in touch with others that they can talk to for help, wiki folk or outside references. Questions should be as specific as possible, as it is easy to give bad/incorrect advice if the advisor does not have all the necessary information. Not sure if this is the right place to put this page...
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''Post new questions at the top of the list''
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[[User:Kathmc|Kathleen McGinness]]
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{{Discussion}}
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==Condensing linear DNA==
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*'''[[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.
 +
 
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==Transduction question==
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*'''[[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.
 +
 
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==Reply==
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*'''[[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.
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*'''[[User:Rachel Davis|Rachel Davis]] 10:03, 31 March 2009 (EDT)''': Thanks so much.
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==E.coli MC4100 thi- genotype==
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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'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===
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*'''[[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:
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<biblio>
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#waldminghaus-2007 pmid=17647020
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</biblio>
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I would be happy to send you any of the mentioned plasmids if they could help you.
 +
 
 +
==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!
 +
 
 +
==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?
 +
 
 +
===Reply===
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*'''[[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==
 +
Does anybody have access (or know about access to) an automatic colony counter? I'm looking for a way to count colonies of different colors. Please email kemd@mit.edu with any information. Thanks.
 +
 
 +
===Reply===
 +
*'''[[User:Rshetty|Reshma]] 11:45, 11 December 2006 (EST)''': You could try emailing the [[Biostuff@mit.edu|biostuff list]] to reach a wider MIT-specific audience.  You may also be able to do something by just taking a photo of your plate and do some sort of image analysis to count colonies.  I've never done this but it shouldn't be that different from what people do to identify cells from microscope images.  Though that might be an overly complex solution to your problem.  Not sure.
 +
 
 +
==Vector LoxP sites flanking two antibiotic marker genes for use in E.coli==
 +
I am in need of a low-copy vector (1 to 2 per cell) which carries two antibiotic marker genes. (preferably one sensitive such rpsL, and
 +
one resistant such at Tetracycline, Chloramphenicol, etc.) and are FLANKED between two LoxP sites so as once i express Cre recombinase i get recombination between the lox sites and removal of the antibiotic sites. I have rpsL gene at the lab, also i have tetracycline. So even if the plasmid doesn';t carry any antibiotic genes, but has LoxP it would be o.k and i may move in with my construct by subcloning between the sites but that would entail more work... :)
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Anyone in a position to help? I would also be very happy,whoever wishes,to collaborate and explain better my underlying project.
 +
email address: acidmucin@yahoo.co.uk
 +
 
 +
===Reply===
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*'''[[User:Rshetty|Reshma]] 08:56, 7 September 2006 (EDT)''': See [[Bacterial artificial chromosomes]] for some options.  Some of the BACs listed there have loxP sites.  But you have to be careful about [[F plasmids| which one you use to really get copy number 1-2]].
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==RBS prediction==
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Is there an online or otherwise easy to use tool for finding RBS sites?
 +
===Reply===
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I've used [http://www.tigr.org/software/genefinding.shtml TIGR's software] to do RBS prediction.  The program is called RBS finder. Also, if you have a specific sequence you want to test, you can use Stormo's work:
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<biblio>
 +
#stormo1994 pmid=8165145
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</biblio>
 +
 
 +
Of couse, this is all for ''E. coli''
 +
--[[User:Skosuri|Sri Kosuri]] 19:22, 23 March 2006 (EST)
==Homologous recombination in ''E. coli''==
==Homologous recombination in ''E. coli''==
If anyone has experience with homologous recombination in ''E. coli'' please check out the [[Standard E. coli Strain for BioBricks]] project and annotate the page with your feedback and comments.  Please include any [[Protocols | protocols]] or [[Materials | materials]] information you might have on the appropriate pages and link to them from the [[Standard E. coli Strain for BioBricks| standard strain project page]] as well.  Any and all advice is appreciated.
If anyone has experience with homologous recombination in ''E. coli'' please check out the [[Standard E. coli Strain for BioBricks]] project and annotate the page with your feedback and comments.  Please include any [[Protocols | protocols]] or [[Materials | materials]] information you might have on the appropriate pages and link to them from the [[Standard E. coli Strain for BioBricks| standard strain project page]] as well.  Any and all advice is appreciated.
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===Reply===
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See [[Standard E. coli Strain for BioBricks]] for comments. Can add more detail later.--[[User:Kathmc|Kathleen]]
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__NOTOC__

Current revision

Post new questions at the top of the list

Condensing linear DNA

  • 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

  • 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

  • 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

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?

Reply

  • 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:
  1. Waldminghaus T, Gaubig LC, and Narberhaus F. . pmid:17647020. PubMed HubMed [waldminghaus-2007]

I would be happy to send you any of the mentioned plasmids if they could help you.

About Trizol

I want to make some homemade Trizol. However I find something very wired in the protocols in Openwetware([[1]]). 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?

Reply

  • 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

Does anybody have access (or know about access to) an automatic colony counter? I'm looking for a way to count colonies of different colors. Please email kemd@mit.edu with any information. Thanks.

Reply

  • Reshma 11:45, 11 December 2006 (EST): You could try emailing the biostuff list to reach a wider MIT-specific audience. You may also be able to do something by just taking a photo of your plate and do some sort of image analysis to count colonies. I've never done this but it shouldn't be that different from what people do to identify cells from microscope images. Though that might be an overly complex solution to your problem. Not sure.

Vector LoxP sites flanking two antibiotic marker genes for use in E.coli

I am in need of a low-copy vector (1 to 2 per cell) which carries two antibiotic marker genes. (preferably one sensitive such rpsL, and one resistant such at Tetracycline, Chloramphenicol, etc.) and are FLANKED between two LoxP sites so as once i express Cre recombinase i get recombination between the lox sites and removal of the antibiotic sites. I have rpsL gene at the lab, also i have tetracycline. So even if the plasmid doesn';t carry any antibiotic genes, but has LoxP it would be o.k and i may move in with my construct by subcloning between the sites but that would entail more work... :) Anyone in a position to help? I would also be very happy,whoever wishes,to collaborate and explain better my underlying project. email address: acidmucin@yahoo.co.uk

Reply

RBS prediction

Is there an online or otherwise easy to use tool for finding RBS sites?

Reply

I've used TIGR's software to do RBS prediction. The program is called RBS finder. Also, if you have a specific sequence you want to test, you can use Stormo's work:

  1. Barrick D, Villanueba K, Childs J, Kalil R, Schneider TD, Lawrence CE, Gold L, and Stormo GD. . pmid:8165145. PubMed HubMed [stormo1994]

Of couse, this is all for E. coli --Sri Kosuri 19:22, 23 March 2006 (EST)

Homologous recombination in E. coli

If anyone has experience with homologous recombination in E. coli please check out the Standard E. coli Strain for BioBricks project and annotate the page with your feedback and comments. Please include any protocols or materials information you might have on the appropriate pages and link to them from the standard strain project page as well. Any and all advice is appreciated.

Reply

See Standard E. coli Strain for BioBricks for comments. Can add more detail later.--Kathleen


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