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restriction analysis

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restriction analysis

Postby siddharthsameer » Sun Jul 15, 2012 6:54 am

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
Last edited by JackBean on Sun Jul 15, 2012 11:20 am, edited 1 time in total.
Reason: made the subject more specific
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Postby JackBean » Sun Jul 15, 2012 11:23 am

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.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby siddharthsameer » Sun Jul 15, 2012 11:55 am

JackBean wrote: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
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Postby JackBean » Sun Jul 15, 2012 7:38 pm

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).
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby siddharthsameer » Sun Jul 15, 2012 8:12 pm

JackBean wrote: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
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Postby JackBean » Sun Jul 15, 2012 11:26 pm

What's your vector name? Do you have sequence or restriction map of it?
http://www.biolib.cz/en/main/

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Postby canalon » Mon Jul 16, 2012 2:27 am

Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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Re:

Postby siddharthsameer » Mon Jul 16, 2012 7:54 am

JackBean wrote: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
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Postby JackBean » Mon Jul 16, 2012 12:16 pm

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).
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: restriction analysis

Postby siddharthsameer » Mon Jul 16, 2012 12:33 pm

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.
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Postby citroenboom » Tue Jul 17, 2012 7:10 am

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.
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Re: restriction analysis

Postby siddharthsameer » Tue Jul 17, 2012 9:14 am

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
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