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    • #16675
      siddharthsameer
      Participant

      hello all
      i am a new student of molecular biology and i have few things to ask i know these are silly questions to be asked but i am unable to find the answer.
      1.how do i interpret the agrose gel result of the PCR and restriction double digestion on the basis of marker.
      i really dont know how to do that,i would be really thankful to you all for helping me with this answer.or if you all can send the link of article or book from where i can read all these would also be appreciated.
      regards
      siddharth sameer

    • #111817
      JackBean
      Participant

      From the gel you simply read sizes of your fragments and then check whether is it the same as expected from in silico/in papiro🙂 prediction of your restriction.
      For that you either
      past your sequence into some program and selects appropriate REs and it will show you the result or
      make a calculation on paper based on positions where your REs cut.

    • #111819
      siddharthsameer
      Participant
      quote JackBean:

      From the gel you simply read sizes of your fragments and then check whether is it the same as expected from in silico/in papiro🙂 prediction of your restriction.
      For that you either
      past your sequence into some program and selects appropriate REs and it will show you the result or
      make a calculation on paper based on positions where your REs cut.

      hello
      Thankd for ur reply.. but can u explain me in expanded form how to do the restriction analysis.. suppose i have a vector of 3.3kb and if i make the double digestion with Ecor1 and Pm1, What is the ideal thing that I should expect and then compare with the gel electrophoresis,please tell me …. please reply me.. thanks a lot in advance regards

    • #111829
      JackBean
      Participant

      You have to find out, where your REs cut. Let’s say it will be in positions 350, 1400 and 3050. Thus you will have fragments 1050 (1400-350), 1650 (3050-1400) and 600 (3300-3050+350).

    • #111831
      siddharthsameer
      Participant
      quote JackBean:

      You have to find out, where your REs cut. Let’s say it will be in positions 350, 1400 and 3050. Thus you will have fragments 1050 (1400-350), 1650 (3050-1400) and 600 (3300-3050+350).

      thanks for the reply but i didnt get anything, rather i got confused kindly explain me brefly.. sorry to trouble you, but i am very new to this field

    • #111832
      JackBean
      Participant

      What’s your vector name? Do you have sequence or restriction map of it?

    • #111834
      canalon
      Participant

      Try to read that:
      http://faculty.plattsburgh.edu/donald.s … stmap.html

    • #111838
      siddharthsameer
      Participant
      quote JackBean:

      What’s your vector name? Do you have sequence or restriction map of it?

      hello
      My nucleotide seuence is of 1428bp and my vector is pPICZalpha A and its about 3,3kb . if i do the restriction digestion with ECOR1 and Kpn1 FOR MY insert (gene of my interest) and my Vector, what is the actual observation I shouls expect… and how do i read or interprate the restriction digestion with molecular marker result.. kindly explain me as I am very new and trubling a lot to find the answer.
      thanking you a lot
      regards

    • #111841
      JackBean
      Participant

      http://products.invitrogen.com/ivgn/product/V19520 here under vector information find your vector and its restriction http://tools.invitrogen.com/content/sfs … a_rest.pdf
      EcoRI cuts at 1209
      KpnI cuts at 1246
      I guess these RE places are introduced at the ends of your insert, right? Thus if you ligate it and re-cut, you will get bands of empty vector (3.3 kbp) and insert (1.4 kbp).

    • #111842
      siddharthsameer
      Participant

      thanks a lot I understood little bit , but in my case as told to me that the ordered nucleotide sequence will be incoroporated with the plasmid and then i have to to subclone it into E.coli TOF competent cell and then i have to plate it select for the colonies and then do the mini prep to get the plasmid and then I have to do the pcr… I really didnt understand How can I do the pcr with the plasmid (Although my gene of intreset in there). but my question is if i do by this process and I do the pcr Should I get the band on 1,4kb AND THEN I EXCISE IT AND CARRYOUT MY FURTHER PROCESS: IF i am right kindly tell me or wrong please guide me with your answer. I would be highly obliged to you, I am unableto understnd this concept.
      regards

    • #111850
      citroenboom
      Participant

      For PCR you need a template = your gene of interest. For this you can use your vector with your gene in it. You design your primers to match the beginning and the end of your gene. Also add the sequence of the restriction sites you want to use, and add GATCTAG to the end of both sides (to give the restriction enzyme space to cut) In this way the rest of the vector is ignored (http://en.wikipedia.org/wiki/Polymerase_chain_reaction) in the process. Use little vector!
      You can purify the product (your gene) from agarose gel or use a PCR purification kit (Qiagen for example).
      Then you have the gene of interest in your hands.
      Add buffer, water and restriction enzyme and leave at 37deg to cut.
      Ligate and transform to TOP10 and you are done.

      For Agarose gel electrophoresis check: http://en.wikipedia.org/wiki/Gel_electrophoresis

      Sorry for using Wiki. It is NOT a source of scientific info, but it can help a lot in understanding stuff and can be a good starting point your search.

    • #111853
      siddharthsameer
      Participant

      hello
      Thanks a lot for your reply one silly doubt once i do the PCR with the vector and then I do the gel electrophoresis , so when I do the gel electrophoresis I should get the band of my desired gene (Suppose my desired gene is of 1428bp) and then I cutthe band and purify it.. is that my cocnecot correct and I shouldget his as a proper result after my pcr.. kindly tell me..
      with regards

    • #111854
      citroenboom
      Participant

      If your PCR is successful (check 5 ul on 0.8% agarose after the run) you can load the whole PCR mix on gel and cut it out. I prefer to use PCR-purification kit, but both works. So, yes, I think your procedure should work fine.

    • #111856
      JackBean
      Participant

      sid: That’s depends, whether is the PCR only to confirm that you have the gene cloned into vector or whether you want to do with the PCR amplicon something subsequently. I would bet on the first option, because pPICZ is expression vector.

    • #111858
      siddharthsameer
      Participant

      hello
      I am about to start and my first step would be the amplification of my desired gene.. and then digestiona nd ligation into expression vector.. so If i start from the first step… thanks you so mucg for clearing my doubts…

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