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cowboy

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  1. Interesting! Thanks for sharing your input... I haven't heard of the RNA world hypothesis before this.
  2. Hi all, I came across an ID50 literature value of #ug/ml for an inhibitor. I wanted to convert this value to M (molar concentration). Is this the right way: # x 10^-6 g/ml divided by molar mass of the inhibitor x 1000 = (ans)M ?
  3. Hmm...is ribosome a protein? I mean any other proteins required for translation, for eg initiation factors, etc I don't know, man... my question sounds a bit stupid.. lol
  4. Sorry, I mean DNA/RNA... But RNA needs other proteins to be translated into proteins as well?
  5. This is something like the chicken and egg story, but it involves DNA. We know that DNA encodes for proteins and proteins are needed for the transcription of DNA. So, which one comes first...DNA or proteins? DNA cannot be transcribed into mRNA (and later translated into proteins) without proteins (RNA polymerases), and proteins cannot be formed without DNA? So, how did the first DNA produce the first protein? :confused:
  6. Does it make any difference? I want a method that is hypothetically correct and can be performed in the lab as well.. ?
  7. Hmm...that is like an entire whole new experiment on its own... hmm.. But thanks a lot for your help. How about this question of mine? Is it really not possible or is it really stupid to do so?
  8. Yeah, you are right. I will get only a smear because there are so many different fragments. So it is really not possible to cut a portion of the smear, elute the DNA and ligate it with the plasmids, portion by portion? Is this what we call creating the gene library? Since bacteria genome is small, it wont take very long, will it? Can you explain more about the two ways you mentioned? I'm not sure about the steps. How do we create a probe for the gene? How do we sequence the amino acid of b-lactamase protein?
  9. Hmm..let say I dont have e.coli b-lactamase nor any other b-lactamase from any other bacteria. If that's the case, I wont be able to use the probing method, is that right? I need to know a small sequence of the gene in order to run a southern blotting. How about this way: - create a gene library by cutting the genomic dna into fragments with restriction enzymes (partial digestion) - run the dna fragments in gel electrophoresis, compare with a known marker - select different fragments and proceed to ligate to plasmid vectors - then everything else same as my post above. Will that work?? My understanding of gene library might be wrong...please do correct me.
  10. After I read some reference books again and again, I think I finally understood the screening process. Anyway, here's my rough plan on how to clone the beta lactamase gene into e.coli. Please see where I've done wrong and also anything else that needed to be added in? Will there be any problems if I actually follow these steps in the lab? Thanks a lot.... Purification of DNA from the isolate - isolate of bacteria is grown in liquid culture medium - isolate is harvested by centrifugation, bacteria will form pellet at the bottom. Medium is poured off. - Break open the cystoplasmic membrane and cell wall by using chemicals or enzyme. Cell lysis. Eg of enzyme: lysozyme, ethylenediamine tetraacetate (EDTA) or both. - Centrifuge to remove digested fractions of cell wall in pellet. Supernatant contains DNA, RNA and protein (called cell extract) - Purify DNA from the cell extract. To remove protein, add organic solvents like phenol and chloroform, protein will be precipitated. Centrifuge to remove the aqueous layer which contains DNA and RNA. To remove RNA, ribonuclease is added – an enzyme which break down RNA into its subunits. - DNA can then be precipitated by adding ethanol. Solution of DNA is added with ethanol and then centrifuged. DNA precipitate will be pellet, and can be redissolved in water. Problems - removal of protein. If there are a lot of protein, treat with proteases which break down proteins into monomers, which is easier to be extracted by phenol Cutting, joining and inserting into plasmid vectors - Since we do not have the knowledge of where beta-lactamase gene is located, DNA treated with a restriction enzyme will produce many fragments, one of which will contain the beta-lactamase gene. For screening purposes, the plasmid used must not have amp-resistance. Therefore, any plasmid with the insert of beta-lactamase gene will have the amp-resistance. Clone gene will be used as the selective marker. - Restriction digestion - So both DNA and plasmid will be treated with the same restriction enzyme to produce the same sticky ends. Sticky ends is preferred, because with the overhangs, complimentary strands can come together more easily and can be ligated. - Plasmid has to be treated with alkaline phophatase to prevent it to recircularised. 5’ phosphate removed from vector, so cannot be ligated together. Can still accept phosphorylated insert. - Make a gene bank by gel electrophoresis. Cut DNA is run in the gel along with a marker and the original uncut DNA. Fragments will be separated according to sizes, cut off gel from each band, and elute DNA from the gel. Proceed to ligation for each band of fragments. - Fragments ligated into vector. Ensure that insert is phosphorylated at one of the 5’ overhang. - PROBLEMS: recircularisation of vector, as solved above. Transform into E.Coli - via electroporation or treatment with chemical. Add iced CaCl2 etc Selection/Screening - Phenotypic selection, first screening – antibiotic to select the cloning vector + XGal , blue white screening, white is the vector of interest. - Second screening – antibiotic to select cloning vector + antibiotic ampicillin to select beta-lactamase gene. - If none is found, repeat for DNA with different fragment size from the genebank.
  11. I'm not asking you to write an essay / or answer in full .... Maybe a rough step-by-step guide like what I've done on my first post will do. I don't understand the part on why we need to know the gene of beta-lactamase. Is it not possible to just clone it into e.coli without knowing the gene? We know that it is amp-resistance, so if e.coli is resistance to amp, the e.coli must have the insert, right? and which screening to use? amp-r or blue/white and which is better? if I could insert the gene into a plasmid which has no amp-resistance and has lacZ operon, I could use both screening method right? Since the plasmid with insert will be amp-resistance and disrupted lacZ (so will appear white in blue/white screening).
  12. Hmmm....I'm still quite confused at some parts. Pardon me as I've just learned this dna cloning only about several days ago. If it's not too difficult, could you guys write down your step by step guide and I'll ask questions from there? Thanks a lot.
  13. First of all, thanks guys for the answers.
  14. Hi everyone...I'm new to this forum. If you think my questions are really dumb, please don't laugh at me...because I'm not that smart... I came across this problem about beta-lactamase cloning. Say, we're given an isolate of bacteria which has beta-lactamase gene and we want to clone beta-lactamase from this bacteria with the help of e.coli. what are the procedures involved? Is this correct?--> - treat the bacteria with restriction enzymes to cut the b-lac gene (but I don't know which enzyme is appropriate - shall I use restr-enzyme like HindIII, BamH1?) - choose a cloning vector. I'm confused at this part too, which plasmid to use? If I am not mistaken, E.coli plasmids contain amp-resistance gene as well like beta-lactamase, so how can i separate the transformed e.coli and the normal e.coli without inserts? -ligate the fragments with cloning vector -transform into e.coli -select e.coli with insert (how to select? can't use phenotypic selection since e.coli without insert has amp-resistance as well? or use blue-white screening perhaps?) Note: please assume that we don't know where the gene for beta-lactamase is.. I'm just so confused at so many things....I hope you all are patient enough to clear my confusion. Thanks a lot.
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