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D.Smalley

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Posts posted by D.Smalley

  1. The most important piece of information given in the question is minimum number of probes. So you work out the possible codons for each amino acid and then choose a 20 nucleotide long segment that has the lowest number of possible codons. This is all to do with degeneracy in the genetic code. When designing DNA probes for an experiment, you must take into account all possibilities, as from the amino acid sequence you have no idea what the actual codon was that coded for, lets say, leucine, as leucine has 6 possible codons.So the first thing to do is to discount those amino acids with the highest no. of possibilities. In this case leucine and arginine. Next look at which amino acids have the lowest: met and 2x trp. From this you should see you only have 2 possibilities to get 20 nucleotides without including Leu or Arg; either start at Pro and end at Phe or start at Trp and end on Val. How do you decide which, when both Pro and Val have 4 options? Well, as you only want 20 nucleotides, the last amino acid of the last codon gets left off. So if we ended on Val then the degenerate nucleotide gets left off, meaning overall we will only have 8 probes.Oh and when writing out the probes you must write out every possibility as shown. I hope that helps :)

  2. I would say to some extent that Marat is correct about the two principles, but I'd say there wasn't too much tension. The idea is that principle one only overrides principle two upon repeated observation of the phenomenon. If under one circumstance you find that '2+3=4' but from thousands of other observations you find 2+3=5, and that this logic applies to many other scenarios e.g. 2+5=7, then, as a skeptic, you logically accept that your observation must be either wrong or not taking into account some other factor.

    With the dinosaur thing, I would say it is always wise to remain skeptical when the only data is someones opinion, no matter how many or who it is from. Empirical data is needed. The discovery of a living dinosaur would be a big deal as our current evidence to counter it is: We have never, in the history of modern man, seen a dinosaur. What did these fisherman do with it? Eat it? Where is it now?

    Besides, if in the 1900s we can find one dinosaur there must be at least hundreds of them to breed for milennia.

  3. I'm confused about something; my lecture notes say (or at least that's how they sound) that if a detergent or phospholipase can remove a protein from the cell membrane, then that protein must be integral. Now, I can understand why detergents and phospholipases can extract transmembrane and lipid-anchored proteins (i.e. integral membrane proteins), but wouldn't they also disrupt the hydrogen bonds and salt bridges of, and thus extract peripheral membrane proteins as well? At least detergents, I'm not sure about phospholipases.

     

    I find it strange that detergents wouldn't also remove peripheral proteins from the membrane in addition to integral proteins, since logically if you disrupt the membrane, you'll also disrupt what is on the surface of the membrane.

     

    Can someone clarify this for me?

     

    Well, this is what I think: The main thing to remember is that integral membrane proteins are in the membrane because they are hydrophobic, peripheral proteins are not in the membrane because they are hydrophilic. Hopefully you'll see where this is going.

    Detergents work on integral memb. proteins because they too are hydrophobic. They won't touch the peripheral ones because they are hydrophilic. The hydrophobicity is the important thing to consider here.

    Hopre that is clear enough :)

  4. Often in the lab, you use restriction enzymes to cut your ligated plasmid to run on a gel, to make sure you have whatever it is you wanted to insert is there. If there's something like that going on, I'd say "aha! an incorrectly ligated vector!"

    Do you know the size of the cDNA insert? If so, that will give you your biggest clue.

     

    Mutation is extremely unlikely.

     

    Do you mean the size from the gel separation? Because by adding up the fragments I got 5.8 for one plasmid and 5.0 for the one missing the duplicated EcoR1 site. But i think you are correct, it must be wrongly inserted as the cDNA is inserted into the EcoR1 site, which means it should be duplicated, appearing at both ends.

  5. Hi guys, i was hoping someone might be able to point me in the right direction on a restriction mapping problem i have to do.

    I have 2 plasmids named 18 and 18L. They are both clones from a pBluescript vector with a cDNA insert, inserted at the MCS. This region was restricted with Apa1 and EcoR1 and then put through an agrose gel separation. From this I have (i think) constructed a restriction map for both. Now, the EcoR1 site is supposed to be replicated at each end, but in the 18L plasmid one of them is missing and the MCS is also shorter: 18 is 5.8Kb and 18L is 5.0Kb with a 'missing' EcoR1 site.

    I have to consider the relationship between these two plasmids. Being as they are the same vector and contain the same cDNA insert, my first thought is some sort of mutation? Or is it just that the cDNA has not been incorporated?

     

    Any help is appreciated :)

  6. I agree with Bobr, Lodish's Molecular Cell Biology is a fantastic textbook and I personally find it more enjoyable to read than Berg. Genes X is a brilliant textbook for genetic classes, so I would highly recommend it for the stuff you said you're doing. It explains every technique very well. I've not looked at Voet & Voet so couldn't comment biggrin.gif

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