Biology Forum Genetics electroporation

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46 replies
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    • #15584
      ZEGSUS
      Participant

      I want to make electroporation but I donit know from which microbΠ΅ to extract DNA and to which to insert DNA.
      Which combination is the most easy?

    • #107164
      JackBean
      Participant

      E.coli -> E.coli

      but I don’t see, why would you do that just to do that.

    • #107203
      ZEGSUS
      Participant

      And is it possible bacillus bulgaricus –>e.coli?

    • #107204
      JackBean
      Participant

      sure, you can do salmon’s DNA into acetobacter. It’s just questionable, whether will the DNA stay or will be degraded or what you want to do with it (but I guess nothing since you don’t care about the organisms πŸ˜€ ).

    • #107209
      ZEGSUS
      Participant

      Well as you know I want to extract DNA from bulgaricus and to insert it in E.coli.The DNA will be in ethanol in the fridge.

    • #107211
      JackBean
      Participant

      yeah, I understood that πŸ˜€ But what DNA will it be? Plasmid or gDNA?

      But I guess you want to clone only small part and insert into some plasmid and express?

    • #107213
      ZEGSUS
      Participant

      I will use and the both type because I haven’t equipment to separate them.No , I will not clone them , I will try directly to insert the DNA into the E.coli.

    • #107214
      JackBean
      Participant

      you have tuo purify the DNA first and that’s were you differentiate between gDNA and plasmid. However, plasmid may be incompatible because of other replication origin. Inserting gDNA doesn’t make sense since bacteria cannot have two chromosomes and thus would one repel probably (even keeping the plasmid without antibiotic selection will be hard).

    • #107215
      ZEGSUS
      Participant

      And how to insert gDNA into the chromosome?

    • #107218
      JackBean
      Participant

      you could use BAC (bacterial artificial chromosome), which has high capacity, but still I’m not sure, whether will it fit whole genome of your bacteria.
      You want to create gDNA library?

    • #107219
      ZEGSUS
      Participant

      And how to use BAC?

    • #107223
      JackBean
      Participant

      I have never worked with BAC, but I guess cut with RE, insert your DNA and transform. Although I don’t know whether electroporation will work for such a big piece.

    • #107224
      ZEGSUS
      Participant

      And what about the plasmids?

    • #107226
      JackBean
      Participant

      these should be fine, but I don’t know whether they have compatible the replication origins πŸ˜‰

    • #107227
      ZEGSUS
      Participant

      So if I want to insert gDNA first I need to cut it and after that to insert it?And how to transform it?

    • #107229
      JackBean
      Participant

      I don’t know how big is the genome, but probably yes, cut, ligate, and transform. I’m not sure what is the size limit for electroporation.

    • #107230
      ZEGSUS
      Participant

      The size ot the lactobacteria is 2.3 Mbp.The limit is <10Kbp.

    • #107236
      JackBean
      Participant

      here http://en.wikipedia.org/wiki/Bacterial_ … chromosome they put the common size of insert into BAC is up to 350 kbp. Thus the number of clones you had to test would be N = [ln(1-P)]/[ln(1-[350/2300])] where P is the probability you will find your gene of interest or whethever are you looking for (99.5%) and 350/2300 is the ratio of insert and whole genome size. As I calculated it the N is ~4, what is odd, since the ration genome/insert is 6.5 πŸ˜† πŸ™„

      I guess best to contact someone who is experienced e.g. someone from some sequencing company/group.

      What is your aim again?

    • #107238
      ZEGSUS
      Participant

      To insert DNA from the bulgaricus in E.coli.

    • #107239
      ZEGSUS
      Participant
      quote JackBean:

      here http://en.wikipedia.org/wiki/Bacterial_ … chromosome they put the common size of insert into BAC is up to 350 kbp. Thus the number of clones you had to test would be N = [ln(1-P)]/[ln(1-[350/2300])] where P is the probability you will find your gene of interest or whethever are you looking for (99.5%) and 350/2300 is the ratio of insert and whole genome size. As I calculated it the N is ~4, what is odd, since the ration genome/insert is 6.5 πŸ˜† πŸ™„

      I guess best to contact someone who is experienced e.g. someone from some sequencing company/group.

      What is your aim again?

      So you mean to cut the gDNA and to attach it to F-plasmids and after that to insert the plasmids into E.coli?

    • #107240
      JackBean
      Participant

      yes, that’s how BACs work

    • #107243
      ZEGSUS
      Participant

      And how to get F-plasmids?

    • #107245
      JackBean
      Participant

      sorry, I have really no idea. But I guess the material providers like Invitrogen or whatever should probably have system for making gDNA library.

    • #107247
      ZEGSUS
      Participant

      http://aem.asm.org/cgi/reprint/60/11/4192.pdf
      Well I guess that is possible to insert "naked" DNA into bacteria.

    • #107252
      JackBean
      Participant

      what do you mean by naked?

    • #107283
      ZEGSUS
      Participant

      gDNA not into f-Plasmid

    • #107284
      JackBean
      Participant
      quote ZEGSUS:

      http://aem.asm.org/cgi/reprint/60/11/4192.pdf
      Well I guess that is possible to insert “naked” DNA into bacteria.

      yeah, but I guess it depends on the species. Agrobacterium is pathogenic bacteria and some of its relatives (I don’t know whether Agrobacterium by itself) can posses linear plasmids. Such bacteria will of course accept "naked" DNA (but still the problem with replication), but it’s questionable how will E.coli do.

    • #107288
      ZEGSUS
      Participant
      quote JackBean:

      quote ZEGSUS:

      http://aem.asm.org/cgi/reprint/60/11/4192.pdf
      Well I guess that is possible to insert “naked” DNA into bacteria.

      yeah, but I guess it depends on the species. Agrobacterium is pathogenic bacteria and some of its relatives (I don’t know whether Agrobacterium by itself) can posses linear plasmids. Such bacteria will of course accept “naked” DNA (but still the problem with replication), but it’s questionable how will E.coli do.

      You mean that the linear DNA cant be save for the future generations?

    • #107291
      JackBean
      Participant

      I really don’t know, whether E. coli is capable of that. Maybe other species would be more sutable for that πŸ˜‰

    • #107305
      canalon
      Participant

      E. coli will not readily take up naked DNA. Other species can, but not E. coli.
      If you are looking for a specific gene from Acetobacter bulgaricus your best bet is indeed to try a BAC. There are many kits commercially available (Google "BAC cloning kit" and your country of residence to find some supplier, or ask your friendly local sales rep) to do just that.
      And no, not all bacteria will accept DNA by electroporation. But in your case that should not be a problem.

    • #107316
      ZEGSUS
      Participant

      In my country the gene engineering is forbidden so there is no suppliers here.And do you have a easy protocol how to insert gDNA into E.coli?

    • #107725
      ZEGSUS
      Participant

      I want to report a little success about my project in electroporation.This is my first prototype of electroporator.I have succeeded to make it with less than 100$.What do you thing?


      Attachments:

    • #107726
      JackBean
      Participant

      seems just like a bunch of wires to me πŸ˜€ (I don’t have much big screen here). Just curious, where are you from, that gene engineering is forbidden? You mean even manipulation with bacteria, right?

    • #107727
      ZEGSUS
      Participant

      http://en.wikipedia.org/wiki/Bulgaria
      Yes even manipulation with bacteria. πŸ™ The "eco organisation" provides a lot of pressure on the government. πŸ™ And the government ban GMO. πŸ‘Ώ

    • #107728
      JackBean
      Participant

      I see. So even Academy is not allowed to work with GM? And I though that EU has strict rules πŸ˜† πŸ™

    • #107732
      ZEGSUS
      Participant

      Yes.But remember -here is Bulgaria.

    • #107755
      JackBean
      Participant

      oh man, you’re part of EU, right? πŸ™„ 😳 My apologies πŸ™

    • #107804
      canalon
      Participant

      I have to say that it is hard to see much from the picture, but if you have managed to generate nice square shocks with the possibility to control the intensity and duration, kudos to you.

    • #107811
      ZEGSUS
      Participant

      Yes , I have.It was difficult but after many attempts I succeeded.

    • #107880
      ZEGSUS
      Participant

      And what about plants?How to insert DNA from plant to plant without plasmids?

    • #107883
      JackBean
      Participant

      for that you would need golden (or from other heavy metal) microparticles with use high pressure to bombard the plant tissue.
      http://www.nepadbiosafety.net/for-regul … ombardment

    • #107885
      ZEGSUS
      Participant

      I know that but is there a chance to insert a genomic DNA with only electricty?

    • #107939
      JackBean
      Participant

      I highly doubt so, because the plant cells have the cell wall, which is pretty good barrier for such transformation. Better way is to use Agrobacterium, which infects plants.
      You want to insert full gDNA again?

    • #107943
      ZEGSUS
      Participant

      Yes.Ok I will use Agrobacterium.Is it possible to transfer gDNA form plant to Agrobacterium?

    • #107959
      JackBean
      Participant

      I though that since you have banned gene manipulation in Bulgaria, you won’t have access to Agrobacterium neither. That’s why I didn’t mention it.
      However, you cannot insert full gDNA using Agrobacterium, since plant genomes consist of several chromosomes and the Agrobacterium transfers only "small" portion from its Ti-plasmid. Nowadays there are available kits for Agrobacterium, which work with two plasmids, thus the inserted DNA may be longer, but it still won’t be full chromosome.
      (and I really don’t see any reason, why to do that)

    • #107989
      ZEGSUS
      Participant

      Well.Lets just say that I have Agrobacterium from somewhere.And thanks about information.

    • #107990
      JackBean
      Participant

      πŸ˜† you’re welcome πŸ™‚

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