Yes but you missed out one important part. NaOH is added to those double stranded DNA fragments to denature them, otherwise complementary probe wont anneal to the target sequence
You forgot the most important step, which is denaturing DNA ( from double strand to single strand ) in order to allow Hybridation between the DNA fragments and our DNA probe... which is followed by a rigorous washing to remove all excess probes that aren't attached.
Explaining the process step by step definitely helped a lot (sth many teachers can't do in 2 hours) but Plz update this video by adding the denaturation and renaturation step and other important details
but what if the gene A was let's just say : CATCCGTA so the probe would be : GTAGGCAT and another gene fragment had a common part like this : CATCCGTAGGT so the first bases are the same is there a chance that the probe would anneal to another gene other than gene A? or even to one where it has a common part of gene A but a shorter fragment ? even if the difference was one extra base ? or the electrophoresis eliminates that possibility? help, please .
If we are studying a certain protein, there are going to be few genes that will fit in exactly with the probe for our DNA of interest. That being said, most prob lengths are more than 100 bases long. That makes it difficult for things to overlap.
It was very helpful though kinda sleepy without any accent to some important points... And yes for the best reply. NaOH or any other alkali treatment would make Dna denature.
I couldnt agree with you more, this is a hibridization in which single stranded cDNA/ RNA will bind to another DNA/ RNA (the one we want to detect) in order to form a hibrid
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>
+Nao Tomori You denature the dna in the gel by soaking the entire gel in NaOH before transferring it to the membrane which makes the DNA single stranded. The probe is also single stranded.
what if it happens that the gene is cut into different fragments which are located in different parts on the gel? Will radiation be detected in several spots then or none of the spots?
+Wayne The enzymes will cut at a specific sequence of nucleotides every time. You will select the enzymes you want to use based on the sequence of your gene and you can ensure that the enzymes will cut in the correct place. It is possible that the enzymes will make a mistake but this won't be very often so the majority of the product will be of the correct length.
The enzyme which is used here is called restriction endonuclease. The main characteristic feature of this particular enzyme is that after searching for a palendromic sequence of bases it will cut at specific locations just away from the centre of the palindromic sequence. It cleave the DNA into fragments and hence it called molecular scissors. Now coming to your question if the enzyme cut the DNA half then there will be error occurred hence it will soon proofreaded by other enzyme such as epsailon0 subunit.
These guy is wrong all over. He has just learnt about Southern Blot probably. Khan academy must Rethink on those selected for teaching these. I believe they are geetting paid good money so please present high quality stuff.
Yes but you missed out one important part. NaOH is added to those double stranded DNA fragments to denature them, otherwise complementary probe wont anneal to the target sequence
saxeli gvari thank you!! i was a bit confused but thanks for clearing it up :)
exactly what i was thinking :D !
Ü
madlobt :)
Yo thank you
You forgot the most important step, which is denaturing DNA ( from double strand to single strand ) in order to allow Hybridation between the DNA fragments and our DNA probe... which is followed by a rigorous washing to remove all excess probes that aren't attached.
What a short great video!My biotech professor confused the f out of everyone! She also took 20 minutes doing that!
Simplified and the best video lecture on southern blotting .
Explaining the process step by step definitely helped a lot (sth many teachers can't do in 2 hours) but Plz update this video by adding the denaturation and renaturation step and other important details
I wanna cry :') that was very helpful thanks I love you
You forgot to mention one important step. After annealing and before exposure to UV, you need to wash away excess radioactive labelled DNA
but what if the gene A was let's just say : CATCCGTA so the probe would be : GTAGGCAT and another gene fragment had a common part like this : CATCCGTAGGT so the first bases are the same is there a chance that the probe would anneal to another gene other than gene A? or even to one where it has a common part of gene A but a shorter fragment ? even if the difference was one extra base ? or the electrophoresis eliminates that possibility? help, please .
If we are studying a certain protein, there are going to be few genes that will fit in exactly with the probe for our DNA of interest. That being said, most prob lengths are more than 100 bases long. That makes it difficult for things to overlap.
so filter is STURDIER than the gel?
It was very helpful though kinda sleepy without any accent to some important points... And yes for the best reply. NaOH or any other alkali treatment would make Dna denature.
Best video on u tube for southern blotting...thanks a lot
how does the DNA probe anneal to the the fragments? after restriction enzymes cut are they single stranded?
Yes the gel is treated with alkali that separates the 2 DNA strands into single strands
I couldnt agree with you more, this is a hibridization in which single stranded cDNA/ RNA will bind to another DNA/ RNA (the one we want to detect) in order to form a hibrid
the probe used is complementary to the targetted gene
You are awesome!!!
nice one @khanacademy. but u missed the NaOH step which is important for denaturing. besides that it was great
tks~~helpful~~
Very very helpful. Thank you
thank youuuuu
Why didn't you denature dsDNA? how can your dna hybridize with probe without denaturation?
Thanks a lot
veey well explained ..thanku sir
Good information
thank you
Excellent!
To the piont .
Really helpful video. Thanks!
Amazing! Thank you so much
Thank you well explained!!!
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>
basically , Southern is used to detect DNA ,Northern is for RNA and Western for polypeptides.
great explanation. love your channel.
Missed blocking step before labeling with probe.
better than my professor's PPT
are piece of DNA=DNA fragments also ds? if yes, how radio-labeled DNA anneals o.O?
+Nao Tomori It has complimentary bases to the DNA fragment so forms hydrogen bonds with it
+Nao Tomori You denature the dna in the gel by soaking the entire gel in NaOH before transferring it to the membrane which makes the DNA single stranded. The probe is also single stranded.
he didn't mention that #3 is referred to as the hybridization step
what if it happens that the gene is cut into different fragments which are located in different parts on the gel?
Will radiation be detected in several spots then or none of the spots?
None of the spots...because our probe can anneal to a complete gene having it's complementary bases,not fragments of it
I can’t understand why we have to separate the dna molecules by gel electrophoresis?.can’t we just add the radio-labeled DNA and then expose to X-ray?
I don't understand this. Where's the more in-depth explanation?
very well explained :) thx buddy
Sooooo......just curious, where are you now?
@@prateekmisra8251 o.o
Thanks , this is so helpful
"Joe electro phoe reese´s"
now you can´t unhear it, you are welcome
🔥
genie.
what if the enzymes cut the gene a in half so the complementary dna can not completely allign?
+Wayne The enzymes will cut at a specific sequence of nucleotides every time. You will select the enzymes you want to use based on the sequence of your gene and you can ensure that the enzymes will cut in the correct place. It is possible that the enzymes will make a mistake but this won't be very often so the majority of the product will be of the correct length.
The enzyme which is used here is called restriction endonuclease. The main characteristic feature of this particular enzyme is that after searching for a palendromic sequence of bases it will cut at specific locations just away from the centre of the palindromic sequence. It cleave the DNA into fragments and hence it called molecular scissors. Now coming to your question if the enzyme cut the DNA half then there will be error occurred hence it will soon proofreaded by other enzyme such as epsailon0 subunit.
How old are you when you are taught this in US?
Evil Assassin 3 months old
The volume is so low
I'd appreciate this video more if I could hear what you're saying!
These guy is wrong all over. He has just learnt about Southern Blot probably. Khan academy must Rethink on those selected for teaching these. I believe they are geetting paid good money so please present high quality stuff.
This is a southern blot video
No. I'm Good