I don't read my notes anymore. I juts watch your videos and I feel confident with upcoming exams. You're such a great mentor. From the Philippines here, Salamat :-)
You are the man, Sir. The amount of work you must have put in to make all these videos is second to none. You are teaching (often times highly) complex matters in a way that makes it very easy to understand for your viewers, and you definitely contribute to a higher educational level among a lot of students, otherwise running their head against the wall, in badly written text books and badly executed lectures at university. Thanks a ton!!
You make the best biochem videos!! I watched your lectures for my biochem course and now studying for my MCAT! your explanations are x1000 clearer than text books and other resources. thank you so much!
I´m form germany, and I liked the explanation very much. Normaly it´s hard for me to follow english science videos, but you talk very clearly and explain the "stuff" often in differnet way, so that even I can understand these difficult processes. Thanks a lot!
I started med school years ago and then letf 1 year later and start over again this year 2022. Why am I saying this? Because now and then there are themes that its special difficult to me to understand and ALWAYS that I look for te solution on AK LECTURES I find it, but its not just a resolution, its a excellent and simple an clear explanation! I have no words enough to say thank you! You're just AMAZING! I wish one day to be as good teacher as you are! You're literally a inspiration for me! Thank you so much!
You take me back man! Been watching you since I first started at uni and now preparing for advanced upcoming exams you're still helping me and lots of students around the world, you're the best!
I really appreciate your work , such a complex topic I was able to understand after struggling over weeks in just 5 min after watching this in 2x, this is how I have written in my notes,. thanks
Your videos are really helpful with my molecular diagnostics class! Our teacher never explains things in detail and it makes it sooo hard to understand anything that she is saying! Thank you for making these! They're a life-saver lol
The point is to isolate the sequence among a large numer of fragments. Maybe you have a sample of dna from a crime scene and you know just a small part of the killer dna, so southern blot can help you find that little fragment among the sample af dna you found in the scene.
genius!!! thanks you speak very well, i´m a latin person and my english is normal jaja.. because the way you speak i could get every word you said!! bravo!
I'm quite confused on what Southern blotting is used for. So just to repeat what you've said - In order for us to perform the southern blotting procedure we need to know the DNA sequence of our desired gene in advance (to make a complementary radio-labeled DNA strand). So two questions about that process: 1) So what exactly southern blotting is used for? Is it used for identifying if an organism has our gene or interest and that's it? What else could we possibly use it for other than that? 2) If indeed we use southern blotting for identifying if an organism has our gene or interest in its genome, why would we need to use the electrophoresis gel for? Can't we just denature the DNA strand and throw it with a radio-labeled probe to see if the DNA strand gets attached to the probe and then we can deduce the DNA strand does indeed have our gene of interest? Why would we need to cleave the DNA strand and run the restriction fragments through electrophoresis if all we search for is the gene of interest? Obviously I could be missing something but I'm quite confused on why exactly we need these additional steps as it seems redundant. Please help?
Regarding the DNA fragments that get transfer into the cellulose sheet, how does DNA denature? For my understanding the DNA in the gel is double-stranded? Maybe the the conditions during the transfer?
It was really awesome. you have taught such a hard topic with clear concepts. I have always feared from Genetics but It was very easy to understand. Thanks for this video😊😊😊
Hello sir. I have a query. Since northern bloating doesn't require denaturation (of ss-RNA), do we need to dip the gel inside alkali solution? Or is it necessary for knocking out certain Nitrogen bases for later radioactive labelling in membrane?
in step 3 after denaturing DNA, why does the denatured DNA attach to the radioactively labeled DNA and not the original complementary strand where it separated from?
hi i have a question.. so in the southern blotting part you said you use radioactively heavy phospjoris atoms to label prob dna. Are 'radioactively heavy phosphorus atoms' intercalating agent?
He didn't mention it but the nitrocellulose paper sits on the gel and the gel sits on a sponge that is soaked in an alkaline solution. This solution is what denatures the DNA into single strand
I think you are just partly right. You denature the DNA duplex using an alkaline solution, but afterwards you usually have to neutralize it, because it wont bind to the membrane at a pH higher than 9.
If anyone could answer that would be cool. How can we put complimentarty of strand of fragment of interest if we did not know which is our fragment of interest? And if we already know that? What's the point?
I don't get it , if you have a radioactive complementary strand why I need to isolate the gene, I can just built on the radioactive strand a complementary strand and amplify the gene I want...( basically my question is where do I get the radioactive strand?)
First of all, the goal of the experiment could be just to find out if there is a complentary gene. This does not sound like much, but back in the 1970s this was more or less the only way to discern eukaryontic genes and is therefore a prelude to todays techniques. The hybridization probe is usally chemical synthesized (eg via the phosphoramidite method) and just a few nucleotides long (20 maybe), so its often much shorter than the DNA you are interested in.
I understand that we can detect complimentary strands using a probe that we built, but if our goal is to extract the gene for testing, why don't we just build complimentary probes (aka: synth our own genes) and use those instead of doing all of this? Am I missing something?
Hey, great vid thank you ! I dont understand why we want to isolate the fragment in the first place if we already have the complementary strand !? Can someone help please :) ? i think im missing something
what is the aspect difference between the three method ? northern, southern and western . i only find a few differences bethween them ? Can you identify the difference>
Thank you very much for the video, helped a lot. I have a question though, and that is: When does the DNA split into to strands ? Is it when it enter the nitrocellulose membranes ?
6:20-6:40 so the DNA probe is complementary okay but the 2 strands are already complemetary to each other why don't they combine and form the helix again but the new added DNA probe does?
Hi there AK lectures, great video - you do a good job explaining the process. I'm wondering - what's the practical application of a southern blot versus a standard PCR amplification & gel electrophoresis genotyping method? Cheers! :)
Sir ur videos are really helpful, thank you for making them😊 but I would like to tell you one thing that some of ur videos have low voice , please correct it if possible.
![BLACK BAG - Official Trailer [HD] - Only in Theaters March 14](http://i.ytimg.com/vi/Du0Xp8WX_7I/mqdefault.jpg)
I'm definitely gonna put your name in my end of studies thesis,wait for it,best teacher ever
Agree
Agreeeeeeeeee
Did you complete your thesis and put his name lol
I don't read my notes anymore. I juts watch your videos and I feel confident with upcoming exams. You're such a great mentor. From the Philippines here, Salamat :-)
Awesome! But I think you should still take notes! Something about the process of writing lets your brain understand and remember concepts better.
Say mahal na mahal kita to him
You are the man, Sir. The amount of work you must have put in to make all these videos is second to none. You are teaching (often times highly) complex matters in a way that makes it very easy to understand for your viewers, and you definitely contribute to a higher educational level among a lot of students, otherwise running their head against the wall, in badly written
text books and badly executed lectures at university. Thanks a ton!!
You make the best biochem videos!! I watched your lectures for my biochem course and now studying for my MCAT! your explanations are x1000 clearer than text books and other resources. thank you so much!
Thanks Mary! happy to help :)
I´m form germany, and I liked the explanation very much. Normaly it´s hard for me to follow english science videos, but you talk very clearly and explain the "stuff" often in differnet way, so that even I can understand these difficult processes. Thanks a lot!
This is so helpful for the MCAT, much easier to understand than any other material I've ever used. Thank you SO much for sharing!!
best lecturer on RUclips. literally saving my life for the MCAT! bless your soul
Sheoll
I started med school years ago and then letf 1 year later and start over again this year 2022. Why am I saying this? Because now and then there are themes that its special difficult to me to understand and ALWAYS that I look for te solution on AK LECTURES I find it, but its not just a resolution, its a excellent and simple an clear explanation! I have no words enough to say thank you! You're just AMAZING!
I wish one day to be as good teacher as you are! You're literally a inspiration for me! Thank you so much!
Your videos are the best. Concise well structured really easy to follow. RUclips teacher of the year award should be yours
You take me back man! Been watching you since I first started at uni and now preparing for advanced upcoming exams you're still helping me and lots of students around the world, you're the best!
no matter how difficult topic it is u never fails to make us understand.. love from Pakistan❤
used your vids for MCAT, still using them for medical school. you are amazing
Difficult topics converts to superbly easy only after watching your video......amazing teacher lots of love...
nice, I've been trying to wrap my head around these processes forever. I have a much better handle on them now, thanks, you're the man.
I really appreciate your work , such a complex topic I was able to understand after struggling over weeks in just 5 min after watching this in 2x, this is how I have written in my notes,. thanks
can never find anyone like you
you are one of kind🙏🙏
Epic lecturer !!!! one of the best on youtube !!
my lecture notes are so complicated that i nolonger read them just watch your videos. cant wait for my distinction. thanks for making the course easy.
Tomorrow I’ll have a biochemistry exam! I’m feeling confident for this topic now. Thanks a lot sir!!
I love you. You got me an A on my last test and you're a great help for the upcoming one. Thank you so much.
This cleared the confusion i had on RNA and DNA separation issues.Thank you bro
Your videos are really helpful with my molecular diagnostics class! Our teacher never explains things in detail and it makes it sooo hard to understand anything that she is saying! Thank you for making these! They're a life-saver lol
You are a great teacher!
It is more easier to understand me to watch your lecture . Thank you for your knowledge sharing
you are very organized and u can explain any difficult topic in a very nice way, thank u so much you are more than amazing ..
Just to add, the dna fragments are denaturated with alkali, before their transfer on to a nitrocellulose filter
Love from Kashmir!!..Allaah bless u nd alwz keep u happy...Such a creative and intelligent teacher...👍👍💙
Thank uhh sir...u r great..u explain the topic in a very easy way..once again thanks a lot Sir🎉
thank you so so much. And now I’m subscribed. Been a great help.
maybe a silly question: if the sequence is known until the point you can build a complementary sequence of it, what is the point of southern blotting?
good question hm
I believe it allows the experimenter to directly isolate the segment of DNA containing the gene from the rest of the DNA (in Southern anyway)
The southern blotting is used to find out whether or not a molecule of dna contains a particular gene ( known)
The point is to isolate the sequence among a large numer of fragments. Maybe you have a sample of dna from a crime scene and you know just a small part of the killer dna, so southern blot can help you find that little fragment among the sample af dna you found in the scene.
THANK YOU. I was having the same bit of confusion.
Great, your english and explication is very clear. Thankyou.
your explanation is amazing...what a good work ..please continue doing that ...its helps a lot
genius!!! thanks you speak very well, i´m a latin person and my english is normal jaja.. because the way you speak i could get every word you said!! bravo!
you are just really great!
The greatest doctor
Thank you from the heart thank you so much ❤️you saved me
well explained...thank you so much will be learning from your videos for the whole project ......excellent work.Satisfied
nicely explained ,thank you my friend for this really nice and useful lecure ,keep up the good work
really helpful with the basics . superb AK
Best lecturer ❤
God bless you 😊
I'm so glad that I found this video! That's a very good explanation, thank you so much :D
I'm quite confused on what Southern blotting is used for. So just to repeat what you've said - In order for us to perform the southern blotting procedure we need to know the DNA sequence of our desired gene in advance (to make a complementary radio-labeled DNA strand). So two questions about that process:
1) So what exactly southern blotting is used for? Is it used for identifying if an organism has our gene or interest and that's it? What else could we possibly use it for other than that?
2) If indeed we use southern blotting for identifying if an organism has our gene or interest in its genome, why would we need to use the electrophoresis gel for? Can't we just denature the DNA strand and throw it with a radio-labeled probe to see if the DNA strand gets attached to the probe and then we can deduce the DNA strand does indeed have our gene of interest? Why would we need to cleave the DNA strand and run the restriction fragments through electrophoresis if all we search for is the gene of interest?
Obviously I could be missing something but I'm quite confused on why exactly we need these additional steps as it seems redundant. Please help?
very outstanding and clear presentation, I understood very easily and I thank you very much!! Please add more videos
this was so helpful as review for my test tomorrow. thank you so much!!
Regarding the DNA fragments that get transfer into the cellulose sheet, how does DNA denature?
For my understanding the DNA in the gel is double-stranded? Maybe the the conditions during the transfer?
+Jacinto De La Cruz Usually NaOH is used to denature the dsDNA
It was really awesome. you have taught such a hard topic with clear concepts. I have always feared from Genetics but It was very easy to understand. Thanks for this video😊😊😊
Thank you for sharing, you simplify this difficult brochure
This man is so helpful
Great lecture, thank u!💖 this lecture exactly help me so much to prepare my uni test🙌
Thanks, I've been learning a lot with your videos, it's really great help! Keep up the good work! :)
Thank you, Great lecture.
Mahbub Rahman you're welcome!
Very simple very easy clear presentation thanks a lot
thank u for helping me during this dumb pandemic
Very simple! Thanks a lot..clearely understood sir
Hello sir.
I have a query.
Since northern bloating doesn't require denaturation (of ss-RNA), do we need to dip the gel inside alkali solution?
Or is it necessary for knocking out certain Nitrogen bases for later radioactive labelling in membrane?
Amazing explanation sir. Well done
This is great! along with the rest of your awesome videos :)
Ahmed Kamal thank you! :)
Why exactly do we need to isolate that DNA fragment if we can make a probe with the exact same sequence in step 4?
That's what I'm so confused about as well
in step 3 after denaturing DNA, why does the denatured DNA attach to the radioactively labeled DNA and not the original complementary strand where it separated from?
you are the reason i'm passing uni
Many thanks, really clear and benefits explanation!
hi i have a question.. so in the southern blotting part you said you use radioactively heavy phospjoris atoms to label prob dna. Are 'radioactively heavy phosphorus atoms' intercalating agent?
this was explained perfectly. thanks!!
How does the double stranded helix become single stranded? The nitrocellulose sheet denatures the double helix? Thanks for such informative lectures!
He didn't mention it but the nitrocellulose paper sits on the gel and the gel sits on a sponge that is soaked in an alkaline solution. This solution is what denatures the DNA into single strand
I think you are just partly right. You denature the DNA duplex using an alkaline solution, but afterwards you usually have to neutralize it, because it wont bind to the membrane at a pH higher than 9.
Do you know what they use to neutralize it?
eg 1.5 M NaCl/0.5 M Tris⋅Cl (pH 7.0)
you explain better than my Prof. lecturer
sending love from egypt to your vedios
This is really good, easy to understand. Thank you so much!! You're awesome !! : ))) Keep up the good work :D
God bless you! ❤️
Are the ssDNA bound to nitrocellulose permanent or can they be removed?
If anyone could answer that would be cool. How can we put complimentarty of strand of fragment of interest if we did not know which is our fragment of interest? And if we already know that? What's the point?
you saved my midterm
Thank you for sharing information🌻
I don't get it , if you have a radioactive complementary strand why I need to isolate the gene, I can just built on the radioactive strand a complementary strand and amplify the gene I want...( basically my question is where do I get the radioactive strand?)
First of all, the goal of the experiment could be just to find out if there is a complentary gene.
This does not sound like much, but back in the 1970s this was more or less the only way to discern eukaryontic genes and is therefore a prelude to todays techniques.
The hybridization probe is usally chemical synthesized (eg via the phosphoramidite method) and just a few nucleotides long (20 maybe), so its often much shorter than the DNA you are interested in.
I understand that we can detect complimentary strands using a probe that we built, but if our goal is to extract the gene for testing, why don't we just build complimentary probes (aka: synth our own genes) and use those instead of doing all of this? Am I missing something?
Hey Chris :)
I just answered the question in the comment section 3 comments down.
does the complementary DNA probe have to match the fragment exactly? Or can it be similar?
fridaymorning yes it has to be it’s complementary
Hey, great vid thank you ! I dont understand why we want to isolate the fragment in the first place if we already have the complementary strand !? Can someone help please :) ? i think im missing something
thank you thank you and thanks gad to find you your way to explain is remarkable and by the way iam from iraqe and its an honor if you answer me
thank you! great lecture!
Thank you , You made it alot easier
what is the aspect difference between the three method ? northern, southern and western .
i only find a few differences bethween them ? Can you identify the difference>
Remember SNOW DROP. Southern- DNA, Northern- RNA, W- Protein
Simply you are soooooo great👍 👌 👍 👌
do you have videos on NGS and other molecular techniques ?
Isn't the probe of Northern blotting suppose to be cDNA?
thanxs doc ... from algeria
Keep up the awesome work!
GoldenSilence333 thanks! will do :)
Thank you very much for the video, helped a lot. I have a question though, and that is: When does the DNA split into to strands ? Is it when it enter the nitrocellulose membranes ?
The first thing that happens in step 2 is the denaturation of the double stranded Dna
What if the restriction enzymes cleaves the gene of interest in between? That is the gene after cleavage becomes a part of two pieces of DNA?
Sanat Mishra since you know the gene you are going to use a specific enzyme that doesnt cut it
now I understood what is sothern blot ...thank you from my heart ❤️❤️🔥🫶❤️❤️🔥
What If the restricion enzym cut the gen of interrest into two. Can the DNA probe still identify the gen fragments?
Sara Hussein This does not happens.
6:20-6:40 so the DNA probe is complementary okay but the 2 strands are already complemetary to each other why don't they combine and form the helix again but the new added DNA probe does?
Because usually you use a very high concentration of the probe so that it will surely bind at least to some of the fragments
Beatrice Colombo Thank you!
Hi there AK lectures, great video - you do a good job explaining the process. I'm wondering - what's the practical application of a southern blot versus a standard PCR amplification & gel electrophoresis genotyping method?
Cheers! :)
Thanks u made it alot easier 👍🏼👍🏼👍🏼
Good explanation
How do we know that during restriction digestion the gene which we want is not getting cleared by the restriction endonuclease?
ConfusedBAKA Gurl We choose that restriction enzyme that doesn’t cleave the gene of interest
@@user-bhavani4 thankyou1
You are amazing!!!
that was clearly explained, thanks:)
I LOVE YOUR VIDEOS!!!! THK U
Sir ur videos are really helpful, thank you for making them😊
but I would like to tell you one thing that some of ur videos have low voice , please correct it if possible.
you are the best!
Nice Work!!