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    • #10607
      thewax
      Participant

      1) How do you get the plasmid ( = how do you isolate the bacterial plasmid from cells) out of the cell? If not from cells (although Campbell and Reece Biology says there are on page 387) where do they come from? 2) Some books say that antibiotic resisting genes are already in the plasmid and some are not. (In Campbell and Reece Biology 7th edition, please refer to page 387 bullet point (1)) If yes, then they naturally have it, but if they naturally have it, then how can they tell it from those that don’t have recombinant DNA (in gene cloning) in the end. If no, how do they get there? If yes, how do people engineer the gene to be placed in the plasmid?

    • #87812
      MrMistery
      Participant

      Please, work through your homework by yourself. People on this website will help you, but you need to show some work first.

    • #87819
      thewax
      Participant
      quote MrMistery:

      Please, work through your homework by yourself. People on this website will help you, but you need to show some work first.

      Please, can’t you tell that this is not homework? And this is not math – how do I "show my work" on something I completely don’t understand?????

    • #87841
      blcr11
      Participant

      The original plasmids were naturally occuring, extra-chromosomal pieces of DNA molecular biologists started to notice right around the same time they began to appreciate the mechanism of bacterial conjugation. There is an OK Wiki article that gives a very very brief overview of plasmids: http://en.wikipedia.org/wiki/Plasmid Another decent source, though dated now, is Gunther Stent’s Molecular Genetics

      The old-style way to isolate plasmids took advantage of two facts: 1) DNA is more stable toward alkali (base) than RNA; and 2) plasmids are much smaller than the bacterial chromosome (although cosmids can get quite large). In very brief outline, you grew the bacterial cells containing the plasmid to high density-usually taking advantage of antibiotic selection; lysing the cells by treatment with alkali; adding solid cesium chloride and spinning the crude extract/CsCl mixture at 35-75,000 rpm for something like 12-24 hours. Under these conditions, cesium chloride forms a density gradient such that, provided you added the correct amount of CsCl, will band the plasmid in one place, and the chromosomal DNA in another place along the gradient. The RNA should have been digested by RNAse as well as by the alkaline treatment and should pellet at the bottom of the gradient, safely out of the way. When you carefully remove the bands, you get fairly pure plasmid DNA, sometimes in very high yields. Now days, you don’t usually do CsCl gradients. The kits are designed to filter or pellet the larger chromosomal DNA, and the columns are much more selective for DNA, and will retain the DNA plasmid while any contaminating RNA and what not gets washes off. When the column is eluted, you get your pure plasmid DNA.

      Most vectors in use today are derived from earlier, natural plasmids, though many have been so highly engineered as to be almost unrecognizable if compared side-by-side with the originals. It is common to piece together different pieces of plasmids from different sources to generate a plasmid for a specific cloning or expression purpose.

    • #87898
      thewax
      Participant

      thanks! πŸ˜€
      can anyone confirm blcr11’s answers?

    • #87899
      thewax
      Participant

      Can someone also explain what blcr11 means (i.e. what RNAse is and what it does, etc.)?

    • #87975
      thewax
      Participant

      can someone please help?

    • #88097
      MrMistery
      Participant

      you can find out what RNase is by googling it.
      And really, you should try to understand the basic processes in biology before attempting to answer difficult questions.

    • #92279
      kk
      Participant

      Let me continue with molecular cloning.

      When you generate N-fusion proteins (our "gene" of interest is fused to the N-term to the reporter gene), does it matter if you leave the Kozak sequence and a start codon in between the two coding regions? If it’s OK how come that there is no reporter protein being produced alone?

      Another question: how many extra amino acids should we allow to have in extra in between our gene of interest and the reporter gene – without problems. I could imagine for example if we had many (5-6-8-10-12..) it could accidentally become an NLS or other signal and our fusion protein will mislocalize.

    • #92310
      MrMistery
      Participant

      if you leave the kozak sequence it shouldn’t matter, you would just end up with a longer linker.

      well it all depends: some proteins may require the N- or C- terminus to be able to move around in order to fold right. However, you’re probably best off starting with 5-10. Also, most of these signal peptides are at the N- or C- terminus, not in the middle of the protein, and the probability of you creating a signal sequence out of chance is really really small.

    • #92336
      Eous
      Participant

      you wouldn’t want to leave the Kozac sequence and start codon in between the two coding sequences just incase the genes should spit, which there is a chance of if you’re trying to splice the gene into a larger sequence or if it is just a plasmid. because then you might have reporters getting made without the protein of interest (false reports) or proteins of interest that are not ‘reported’. the chance of them splitting is not always significant, but if they do it could seriously mess things up for you. if there is only one kozac and start codon, then then split would nullify one gene (the one that no longer has the stuff to start with) so you would only have to worry about one of your problematic situations. i think you’d be better off with genes of interest getting transcribed without reporters cause usually you’re segretating things afterwards based on the reporters (like trying to grow colonies of bacteria based on resistances and such) so if you start with the gene of interest and then the reporter, you won’t have incorrect cells in your batch). at least, that’s how i see it.

    • #92341
      kk
      Participant

      Thank you. Interesting argument on the extra-Kozak and start codon…

      So is it even better to leave a few extra amino acids as for a linker in a fusion protein? First I thought the best is if the reporter sequence and "our protein" is right next to each other. It makes sense though that extra amino acids would allow correct folding.

    • #92818
      vidya790
      Participant

      Hi RNAse is nothing but Ribonuclease which actually cleaves a phosphodiester bond between any two ribonucleotides and also degrades RNA into smaller components which is mostly helpful in prokaryotic processes and play a important role in many biological processes.

    • #93428
      kk
      Participant

      I have a few besic questions due to I am simply lazy to look up old lecture books.

      Before ligation of an insert into a terget vector, the target vector is treated with phosphatase, to remove WHICH phosphate group? The one at the 3′-end, 5′-end, or both?

      When a restriction endonuclease cuts a double stranded DNA, which newly formed end will retain the phosphate group from the phosphodiester bond? Is it the 3′ or the 5′-end?

      THANKS A LOT!!!

    • #93489
      JackBean
      Participant
      quote kk:

      I have a few besic questions due to I am simply lazy to look up old lecture books.

      You shouldn’t say that πŸ˜€

      quote kk:

      Before ligation of an insert into a terget vector, the target vector is treated with phosphatase, to remove WHICH phosphate group? The one at the 3′-end, 5′-end, or both?

      There is only one, so pick, which one is it πŸ˜‰

      quote kk:

      When a restriction endonuclease cuts a double stranded DNA, which newly formed end will retain the phosphate group from the phosphodiester bond? Is it the 3′ or the 5′-end?

      See above πŸ˜‰

      Regarding the Kozak’ sequence, the note was right, you should not leave it in the midlle of transcript, otherwise transcription will statr also there (e.g. I saw it right yesterday in Invitrogen’s manual;)

      Regarding spacing AAs, depends on where in the structure are the ends. If they are exposed to surface, any (=zero) AAs can work, on the other hand, if they will be inside, it may cause misfolding, if there were not enough of spacing AAs πŸ˜‰

    • #93788
      kk
      Participant

      Thanks JackBean! I am aware that there is one phosphate per nucleotide. I meant:

      Restriction endonucleases: when they cut which newly formed end retains the P group, the 3′ or the 5′?

      Shrimp alkaline phosphatase: from which end does it have the ability to remove a P group, the 3′ or the 5′ or both?

      And actually one more thing I don’t get: at the NEB website, for BamHI it says "Heat inactivation: No". What does it mean? No way to inactivate it? πŸ™‚ No need to inactivate it – why?

    • #93794
      JackBean
      Participant

      Shimp AP? What is that? πŸ˜€
      just 5 seconds to type it and… tradaaa
      http://www.fermentas.com/catalog/modify … phosph.htm
      Description
      The Shrimp Alkaline Phosphatase (SAP) catalyzes the release of 5′- and 3′-phosphate groups from DNA, RNA and nucleotides. Also this enzyme can remove phosphate groups from proteins.

      (usually the phosphatases don’t care much from where they are removing the phosphate, just do it πŸ˜‰

      Indicates whether or not the enzyme can be heat inactivated. Enzymes are first tested by incubation at 65Β°C for 20 minutes; any enzyme not inactivated at 65Β°C is then tested byincubation at 80Β°C for 20 minutes.

    • #93822
      MrMistery
      Participant

      this shrimp alkaline phosphatase is just a type of CIP. They now have a million of them, but they do all the same thing. Personally I rarely need to use it. My problem is usually lack of colonies not high background.

      @kk
      obviously if you heat a protein enough it will get inactivated due to misfolding, but if you boil DNA it will also cause nicks. Generally you shouldn’t put the DNA at above 85 degrees celsius. NEB tests their enzymes at 65 and 80, like JackBean said http://www.neb.com/nebecomm/tech_refere … vation.asp

    • #106231
      kk
      Participant

      My current problem:

      I want to ligate synthetic oligo to a plasmid (I ordered 2 single stranded DNA oligos, which are reverse complementary of each other except their ends, I hybridized the two oligos, now I have an insert with both ends sticky).

      Should I use alkaline phosphatase (CIP, SAP) to remove 5′-phosphate from the digested plasmid vector (to prevent self ligation)? In other words: does the synthetic oligo contain 5′-phosphate (which will then be needed for ligation)?

    • #115039
      mohajeri
      Participant

      I want to clone a human gene in pET-21a, and transfer to E.coli. I clone the gene in Nde 1 site that it’s sequence is CA/TATG, My question, is The ATG of Nde1 act as a start codon in result I must delete The ATG of my sequence or I can use the ATG of vector in Nde1 site as a start codon? (NOTE: My gene is eukaryotic)
      Is there anyone make a sugestion?

    • #115040
      JackBean
      Participant

      You replace one ATG with another, don’t you?

      However if I were you, I’d be rather concerned about the start codon of the N-terminal fusion.

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