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Thread: Looking for female and male nepenthes leaves for sexing experiment

  1. #25
    pokie22's Avatar
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    Quote Originally Posted by amphirion View Post
    @pokie: using ePCR or traditional?

    for DNA analysis will you be running the DNA through a next gen sequencer or will you be doing the good ol' DNA gel electrophoresis?
    The PCR product will have to be cloned into a plasmid for sequencing to occur. The PCR method is using RAPD primers which are short primers that will bind multiple places in the genome. In the paper, a single primer gave this "male specific band" thus precluding sequencing off the PCR product. Cloning a PCR product can be done via a number of ways, none of which are cheap. I will probably use the Gibson assembly or one of the PCR topoisomerase plasmids from Invitrogen. Once the PCR piece has been cloned into the plasmid, the DNA insert can be sequenced directionally. We don't sequence in house. The company that I like to use probably uses the traditional Sanger method.

    If there is no band to be seen, and the paper is a fluke, this will require more effort on my part as I will have to do some bioinformatics.

  2. #26
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    Quote Originally Posted by Heli View Post



    so in layman's terms you are trying to find the coding in the DNA that determines the sex of the plant by amplifying it through cycles?
    no she meant she needs enough DNA to make analysis meaningful, so if you send a tiny amount it will have to be replicated many times to get the minimum amount. the more times it's replicated the greater the probability that something can go wrong, thereby decreasing confidence in the results. so don't send a tiny piece of a leaf, sacrifice a whole new leaf.

  3. #27
    Sphagnum Guru Wire Man's Avatar
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    Here's what I can send;

    Female:
    -N. khasiana
    -N. alata
    -N. 'Red Dragon'

    Male:
    -N. x mixta
    -N. 'Lumpy Space Princess'
    -N. maxima Dwarf Form

    Unknown:
    -N. lavicola SG
    -N. naga SG
    -N. maxima 'Lake Poso' SG
    -N. veitchii SG
    -N. fusca SG
    -N. 'Caladium' SG
    -N. 'Nile' SG
    -N. 'Dormouse' SG
    -N. 'Lorraine' SG
    -N. ventricosa SG

  4. #28
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    Even though it appears that your needs have been met, I'll add these since two of the 'provens' are of the same species.

    Male
    - N. aristolochioides - flowered

    Female
    - N. aristolochioides - flowered

    Unknown
    - N. hamata - SG

    Please PM me if you'd like these leaves.
    All the best,
    Ron
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  5. #29
    i dont do pots. amphirion's Avatar
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    Quote Originally Posted by pokie22 View Post
    The PCR product will have to be cloned into a plasmid for sequencing to occur. The PCR method is using RAPD primers which are short primers that will bind multiple places in the genome. In the paper, a single primer gave this "male specific band" thus precluding sequencing off the PCR product. Cloning a PCR product can be done via a number of ways, none of which are cheap. I will probably use the Gibson assembly or one of the PCR topoisomerase plasmids from Invitrogen. Once the PCR piece has been cloned into the plasmid, the DNA insert can be sequenced directionally. We don't sequence in house. The company that I like to use probably uses the traditional Sanger method.

    If there is no band to be seen, and the paper is a fluke, this will require more effort on my part as I will have to do some bioinformatics.
    sanger's method sounds definitely like a pretty penny, but it offers the best accuracy in reasonable time.

    so basically, if I follow, you plan on replicating the entire genome to find that specific male sequence. would you then isolate and PCR that sequence to utilize as a future template? is it possible to create restriction enzymes that recognize that sequence so you dont have to repeat the sanger process again? i could see you utilizing that template as a standard to run against future inquiries. isolate DNA from new nep sample, run restriction enzymes, purify, PCR, then run a gel so you wouldn't need to sequence any longer to determine the sex.

    14 samples already @ roughly $2400 a pop.
    " You keep using that word. I do not think it means what you think it means." -Inigo Montoya
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  6. #30
    pokie22's Avatar
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    Quote Originally Posted by amphirion View Post
    sanger's method sounds definitely like a pretty penny, but it offers the best accuracy in reasonable time.

    so basically, if I follow, you plan on replicating the entire genome to find that specific male sequence. would you then isolate and PCR that sequence to utilize as a future template? is it possible to create restriction enzymes that recognize that sequence so you dont have to repeat the sanger process again? i could see you utilizing that template as a standard to run against future inquiries. isolate DNA from new nep sample, run restriction enzymes, purify, PCR, then run a gel so you wouldn't need to sequence any longer to determine the sex.

    14 samples already @ roughly $2400 a pop.
    No, the primer amplifies just the piece of interest. Amplifying the entire genome is not possible because the polymerase is limited by its processivity. With the most processive DNA polymerases, 6kb has been my best. No one runs gels these days. Sequencing is cheap - I don't know where you got that number from - $10/run. Each run usually gives 700-800bp of good sequence. Once one has the sequence in hand, then a unique specific assay can me made. This is like the ground work for the real assay. The chicken or the egg......

    The creation of novel restriction enzymes is an entirely different matter. And in response to your earlier comment about lucerative endeavors, that is one of them. Creation of novel molecular tools entails lots of effort and usually some insight. Those are my kind of projects. This is simply a favor to a friend. I had placed this pet project on the level of weekend grocery shopping.......

  7. #31
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    Quote Originally Posted by pokie22 View Post
    I had placed this pet project on the level of weekend grocery shopping.......
    that high? this seems like wipe-up-a-spill type of stuff to me...

  8. #32
    i dont do pots. amphirion's Avatar
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    ah. i see. $2400 a pop comes from the cost of a 1 million bp run. 700-800 pb is definitely not 1 million
    looking forward to your findings! thanks so much taking this on.
    " You keep using that word. I do not think it means what you think it means." -Inigo Montoya
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