Southern blotting
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- เผยแพร่เมื่อ 7 มิ.ย. 2016
- Southern blotting - This lecture explains about the southern blotting procedure and technique to identify specific DNA fragment from a mixture of DNA molecules separated by the Agarose gel electrophoresis. Southern blotting step by step explanation is provided to understand the procedure of the DNA separation and identification. Southern blotting application is also explained in this video.
Southern blotting consists of three different stages -
1. First is the isolation of DNA from the target cell.
2. Then the treatment of restriction digestion of the DNA to generate DNA fragments
3. Then the separation of the DNA fragments using Agarose gel electrophoresis
4. Transfer of the Agarose gel DNA bands to a nitrocellulose Membrane filter. This filter paper after the DNA transfer is known as southern blot.
5. Then the use of complementary DNA probe to bind with the target DNA that will indicate the presence of target sequence by radioactivity or colorize trick expression. Southern blotting was invented by EM Southern on 1975 at it was named after him to identify DNA using blotting technique.
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Thank you for watching southern blotting lecture.
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I'm a student of biotechnology. I found that ur 100time's better than my prof. Thank you sir. May god bless you.
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I also
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Very comprehensive.
I am a doctor and you have very well explained about all blotting methods.Thank you
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Same here. He is so on point
I think the lab technicians would know more about blotting than doctors? Because it's literally what they do everyday!
Thank you for making this video. You've explained this way better than my prof!
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Student from Thailand.
Great video! I have Southern Blotting as the subject for my biology presentation, and this is the only thing that helped me understand what Southern Blotting is. Thank you!
Glad to hear that you're getting benefit from my lectures
i have been watching your vids since many years now.. and you have really improved in ur explanations... ur older vids are nice as well.. but the newer ones are even better.. i prefer this format over the older s.blot video!
Southern blotting was a foreign concept to me before I watched this video. Thank you, Shamu....as usual!
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The video made me understand southern blotting very easily. Off to watch northern blotting! Looking forward to more of your content!
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thank you sir... your video has actually cleared all my concepts... this is the first time when i actually understood the exact meaning of probe n need of using nitro cellulose filter..... thanks a lot sir... god bless u.
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Ameen
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No professors explained what southern blotting really is. So you are the best professor for me. Thank you 😊
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This video was so helpful! It was the best explanation on Southern blotting I have ever seen. Thank you so much!
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@@shomusbiologyofficial Welcome sir
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Denaturation of fragmented dsDNA is done before transferring to NItro cellulose membrane. Otherwise how this DNA will attach to the membrane.
U r right it should have done before tranfering into nitogenous paper
Hey can you tell me why those ss dna dont bind to each other again before binding to probe ???
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You made this much easier to understand, God bless you sir.
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Why do you need alkaline buffer? What would happen if I used acidic buffer, water or Histidine? Why does it HAVE to be alkaline buffer for the imprint to work?
Thank you ^.^ I understand now
dna is negatively charged(phosphate group). alkaline buffer is more basic (ready to share an H+) and acidic buffer is ready to take an H+. in this reaction dna is the acid and is reacting with the basic buffer... this is my thinking..and reference from my orgo chem class... i think the phosphate group which consists of P(-)H3, where the phosphate atom is negative, would give away other H if combined with something more acidic or some compound wanting a H more. Causing the dna to become more reactive.
Acids contain excess H+ ions, which bind quickly to the phosphate (PO4)- backbone and cancel out the negative charge. Without charge, DNA can't migrate because it won't be attracted to the (+) electrode. The alkaline buffer does the opposite, neutralizing any H+ (or Ca2+, Mg2+, or other + ion) by pulling them off the DNA, into solution. Alkaline means excess OH-, so by dissolving any (+) ions, it makes sure that the DNA will be (-) and be attracted to the positive electrode (allowing migration and development of bands).
But what if the restriction endonuclease (DNase I ) cuts in between the target DNA , Then how our probe will bind to target DNA?
megha sharma the restriction endonuclease cuts in specific places only. If you know the sequence of the target dna then you can control which endonuclease to use to cut in what location.
creo que la enzima de restricción solo corta en ciertas partes.
Very nice explanation.
Very much easy to understand.
Thanks
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Thank u so much.... Really.. I was so confused with it... But now it's clear.. Ur the best 👍💯..
Sir I am have a doubt.
How do we know that the DNA of the required sequence is 100% present?
Cause we have prepared complementary probe of that sequence right? How do we know that's sequence is present? :/
Also we can do sequencing to know whether it's there or not? I just don't understand.
And also sir can you please explain regarding restriction mapping and restriction digestion. Cause I've watch all of those videos but you haven't mentioned about the partial digest double digest and all. And no one can explain better than you its a requesttttttttttt. And i am in th ir d year biotechnology can you suggest reference books for RECOMBINANT DNA TECHNOLOGY.
actually as far as i know we do sB in order to confirm the gene of interest.
we have gene banks where we have sequenced the genome
and we make the probes against the segments whose secquuences we have already done
and because of hybridization technique ie probes we can diagnose
I hope u have got it
this fanda is diagnosis ☺️
Don't we use nylon membrane in Southern Blotting and nitrocellulose in Western Blotting? Im confused..
Aishwarya D my same initial thought . can anybody explain ? I'm confused
Aishwarya D either nitrocellulose or nylon membrane can be used in both the technique .
both can be used
Thank you so much, you are one of the best Biologist
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Beautiful video you made about southern blotting
Thank you so much for the detailed teaching methodology of yours....I am much benifited...
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Very informational video, thank you. Well explained!!!
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I clearly understood southern blotting from Ur video thank you 😊 so much
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Your lecture help me to understand boltting ...thank you Soo much
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bro u r a thousand time better than professors in my university
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Thank you so much
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you have a brilliant way of explaining thanks a lot
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Thank you for making us understand it this easily👍
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Thank you for excellent class