What if the two single-strand have the same mass? How can you make a difference with gel electrophoresis? For example: 5' -AGCCATTG-3' and 3'-TCGGTAAC-5'
@@mauktiktiwari4589 I know that, but if you have this piece of DNA and you subsequently denature it by heating, you can't distinguish them from each other on a gel because they have the same amount of purines in both strands. They even have the same mass, so their "stripes" on the gel will overlap. If you can't distinguish one strand from the other, you will get an invalid result on the gel after Gilbert sequencing. For example: you could get stripes in every lane. This method is quite obvious and clear to me but for the piece of DNA I proposed, it doesn't apply in my point of view.
Yes in your case the strands will not have a difference singe A+G is the same....so if one single band is obtained the scientist will increase or decrease the banding pattern so as to obtain to bands on a gel which will allow them to excise and extract the ssDNA to proceed.
I have to question please. 1-How we make a probe in the first place? It’s an unknowing sequence so how we know the sequence to make a primer. As far as I understood after searching, is it that first we isolate the DNA from the cell in tube with a plasmid then randomly add multiple RE(Restriction enzymes), after the cut happened to both DNA and PLASMID. For example, the EcoR1 will cut at its restriction site on both DNA and Plasmid then we can add a DNA ligand and part of DNA will be in the plasmid. We do know the sequence of the plasmid so we use a primer for the plasmid starts before the restriction enzyme, then we can know part of the targeted DNA. This new information about the sequence will help up make a primer to the targeted DNA and then we will perform sequencing method and we know the sequence. Is the way I describe it correct? Also, is it possible that many they will be no RE? Then what we can do? 2- Why we need to radiolabeled the DNA phosphate beginning at 5’? I’m hoping u see this and reply to me.
5' end of one stand is labelled so ahet autoradiography you can see only that strand. We only need to track the development of one stand from one direction to the next.
@@BioMagica It did help a lot, thank u so much. I’ve been looking for these answers and I couldn’t find anyone to correct or help me except u I appreciate thank u sooooo much. Also, please if you could tell what are the references u usually read to help more about this subject. And sorry if I bothered u❤️❤️.
If you want to find the first base, the best method would be to sequence both the strands, so the last base in each strand would represent the first base in the complementary strand. Or you could use overlapping fragments. Or excise the 5' nucleotide, and then identify through chromatography.
04:26 The pH for modified G specific cleavage is alkaline and for either A/G is acidic (formic acid based).
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What if the two single-strand have the same mass? How can you make a difference with gel electrophoresis? For example: 5' -AGCCATTG-3' and 3'-TCGGTAAC-5'
You dont need to do the sequencing of 3'-5' as it is just complimentary
@@mauktiktiwari4589 I know that, but if you have this piece of DNA and you subsequently denature it by heating, you can't distinguish them from each other on a gel because they have the same amount of purines in both strands. They even have the same mass, so their "stripes" on the gel will overlap. If you can't distinguish one strand from the other, you will get an invalid result on the gel after Gilbert sequencing. For example: you could get stripes in every lane. This method is quite obvious and clear to me but for the piece of DNA I proposed, it doesn't apply in my point of view.
Yes in your case the strands will not have a difference singe A+G is the same....so if one single band is obtained the scientist will increase or decrease the banding pattern so as to obtain to bands on a gel which will allow them to excise and extract the ssDNA to proceed.
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Happy to help 😊😊😊
I have to question please.
1-How we make a probe in the first place? It’s an unknowing sequence so how we know the sequence to make a primer.
As far as I understood after searching, is it that first we isolate the DNA from the cell in tube with a plasmid then randomly add multiple RE(Restriction enzymes), after the cut happened to both DNA and PLASMID. For example, the EcoR1 will cut at its restriction site on both DNA and Plasmid then we can add a DNA ligand and part of DNA will be in the plasmid. We do know the sequence of the plasmid so we use a primer for the plasmid starts before the restriction enzyme, then we can know part of the targeted DNA. This new information about the sequence will help up make a primer to the targeted DNA and then we will perform sequencing method and we know the sequence. Is the way I describe it correct? Also, is it possible that many they will be no RE? Then what we can do?
2- Why we need to radiolabeled the DNA phosphate beginning at 5’?
I’m hoping u see this and reply to me.
Yes that is one way of creating primers the one you described
Mostly RE sites are available.. There are many restrictions enzymes
5' end of one stand is labelled so ahet autoradiography you can see only that strand. We only need to track the development of one stand from one direction to the next.
Hope it helps. Do let me know if you need more clarification
@@BioMagica It did help a lot, thank u so much. I’ve been looking for these answers and I couldn’t find anyone to correct or help me except u I appreciate thank u sooooo much. Also, please if you could tell what are the references u usually read to help more about this subject. And sorry if I bothered u❤️❤️.
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@@BioMagica off course
Why can't the 1st base in sequence identified by this method?
If you want to find the first base, the best method would be to sequence both the strands, so the last base in each strand would represent the first base in the complementary strand. Or you could use overlapping fragments. Or excise the 5' nucleotide, and then identify through chromatography.
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