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Transformation & Minimal Plates


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#1 Chris Max Planck

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Posted 14 June 2011 - 02:32 AM

Hi !

I would like to post my personal experience regarding the use of minimal plates at least in my case that I would like to perform an in vivo complementation assay. I have generated a library of mutants with error prone PCR and also an E.coli strain with certain deletions for my purpose for the screening method. For that reason, I have to use minimal agar plates and not with full LB medium. What I have experienced up til now, is that after transformation of my e.coli competent cells with my library of mutants, I observe a kind of precipitation on the minimal plates. At the beginning I was not sure if this happens to the cells of just to the culture medium, because I use normal LB medium for the recovery of the cells after transformation. This kind of precipitation which seems like an aggregation is normally distributed around the minimal plate, and of course this does not happen in case of normal LB plates. Additionally, the cell growth is quite slow, I just observe cell colonies after 4 days, which is normal in my opinion. I would like to ask you if anybody of you has worked with such kind of experiments and minimal plates and he/she might have observed that. Is there any special protocol for transformation just for the case that the cells will be streaked in minimal plates and not in LB pates? Do you have any suggestions for the minimal plates synthesis in order to make them more highly nutrient? I just use an M9 recipe and also some micronutrients. Any ideas for boosting the cell growth? (OK I know to provide all the amino acids e.t.c but it is quite expensive). Any tipps concerning minimal plates, transformation for that purpose, cloning and anything else you consider useful, are extremely welcome.
Thank you very much for your time and your patience and of course for your help in advance.



Best,


Chris

#2 phage434

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Posted 14 June 2011 - 03:40 AM

I'm not sure why you are growing on minimal plates. If it is to get reproducible conditions, and you aren't looking for specific phenotypes, you might want to look at EZ-Rich defined medium, which is a chemically defined rich medium for E. coli. In general, E. coli lab strains need thiamine in the minimal medium, which would be a good place to start. Check the strain phenotype for what else your strain might need.

#3 Chris Max Planck

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Posted 14 June 2011 - 05:03 AM

Dear Phage434,

thank you very much for your reply. I have to underline that minimal plates are absolutely necessary because as you mentioned, I am looking for phenotype. In other words, to be more specific I am looking for colonies which will express my ligated gene of asparaginase and this enzyme after hydrolysis of asparagine, will provide to the cells the availability of aspartic acid (product of enzyme catalysis) in order to grow. In that way I will have a direct mean to monitor and correlate cell growth with high enzymic catalysis of my mutants since the availability of aspartic acid will be proportional to the cell growth. Furthermore I have deleted the 5 genes which contribute to the synthesis of aspartic acid inside the cell, which means that lack of aspartic acid results in cell death. Summarizing, the minimal medium is the only solution for me. I have seen what you mentioned regarding the thiamine, but it is not true for all strains. It must be a very specific strain which requires the presence of thiamine. But I will consider it the next time I will prepare my plates since you also suggested that. In case you have something else I would appreciate your help. Be well and thank you again Phage434.....

Best,

Chris

#4 Evanescence

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Posted 17 June 2011 - 01:04 AM

Hi Christ,

I was using M9 minimal medium (Sambrook handbook) with thiamine as supplement to grow E.coli JM109, i agree with you if you use different strain u need to check the genotype..
Like your case i am deleting certain gene which need me to use minimal medium, so i decided to use M9 medium glucose and thiamine...So what you need to do now, check the back ground of your strain...Also maybe you can add some nitrogen compound to support the cell growth..Wish you luck:)

#5 Chris Max Planck

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Posted 18 June 2011 - 12:13 PM

Hi Christ,

I was using M9 minimal medium (Sambrook handbook) with thiamine as supplement to grow E.coli JM109, i agree with you if you use different strain u need to check the genotype..
Like your case i am deleting certain gene which need me to use minimal medium, so i decided to use M9 medium glucose and thiamine...So what you need to do now, check the back ground of your strain...Also maybe you can add some nitrogen compound to support the cell growth..Wish you luck:)



Thanks Evanescence for your interest and your reply..... It seems that we are running in parallel a similar experiment. I would like to describe you what is my main concern about this trials. As I said at the beginning , I transform my strain with the gene deletions with plasmid of interest following the typical traditional protocol for transformation. Then I use this mixture in order to streak directly my minimal plates. My observation is, that after the addition of the transformation mixture onto the minimal plates, and once the plates are dried, there is a formation of a kind of precipitation on the plates. I do not know if this is due to the different composition of the LB I used for the recovery of the cells after transformation and that of the minimal plates or I am doing something wrong....I do detect colonies after 3-4 days at 37 degrees, but these colonies do not look like these ones on normal LB plates. They look like they are covered by this precipitant I mentioned above. Have you ever observed that phenomenon? Do you follow a different strategy for transformation and/or streaking of minimal plates? I would appreciate if you could share with me such information. Thank you very much in advance for your time and your help.

#6 Evanescence

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Posted 29 June 2011 - 04:30 AM

Christ, it is good reference i still using now...Sambrook handbook complete with all guides u need...but i dont know if it still possible to still get it now, i am get a copy from my lab..it's old one still can read though!
lol




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