not sure if you already found out. IIRC phenol is a bit soluble in water. chloroform prevents that. and isoamyl alcohol prevents bubbling, which i'm not so sure what that will affect. perhaps visibility. using phenol n chloroform alone would do in ratio 1:1. repetition needed to make sure you have removed as much as protein. usually people do 3 cycles of purification before precipitating DNA
A very good movie, however, all mixing steps were performed by pipetting and this could be problematic for DNA sharing or damage as we know that gDNA is a fragile component. I prefer the up side down method to avoid such sharing.
I have a little question. Why are you transferring Phenol:Chloroform:Isoamyl in an open area. This solution is toxic and carcinogenic so it must be done under fumehood. Thanks.
Jit Goria lol this is an instructional video and to me pretty much looks like it’s done safely. You really can’t see the background or anything because everything is done close up. Just like most other instructional videos made by companies out there.
Because the eppendorf while centrifugation won't reach an horizontal state. The angle is locked so that it doesn't reach that state. Meanwhile the acceleration vector is horizontal(because of rotation) , therefore your DNA will stack up on the side of your tube. This way it will tend to "glue" to the tube once precipitated instead of flowing with the sumernatant while you eliminate it. Sorry for bad English.
This is because gram +ve bacteria have thick cell walls and hence are more difficult to deal with as compared to gram -ve bacteria. I am dealing with the very same problem.
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Thank you so much! This video remains super helpful after 10 years!
not sure if you already found out. IIRC phenol is a bit soluble in water. chloroform prevents that. and isoamyl alcohol prevents bubbling, which i'm not so sure what that will affect. perhaps visibility. using phenol n chloroform alone would do in ratio 1:1. repetition needed to make sure you have removed as much as protein. usually people do 3 cycles of purification before precipitating DNA
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A very good movie, however, all mixing steps were performed by pipetting and this could be problematic for DNA sharing or damage as we know that gDNA is a fragile component. I prefer the up side down method to avoid such sharing.
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*shearing of DNA. Yes, true. Inversion for gentle mixing is best.
I have a little question. Why are you transferring Phenol:Chloroform:Isoamyl in an open area. This solution is toxic and carcinogenic so it must be done under fumehood. Thanks.
Right
It's indian style...what can I say..
Jit Goria lol this is an instructional video and to me pretty much looks like it’s done safely. You really can’t see the background or anything because everything is done close up. Just like most other instructional videos made by companies out there.
You should also mention the reasons that why,for what purpose we use particular chemicals,.. Otherwise great tutorial.
How much volume do you use to resuspend DNA in TE buffer?
Can I ask something? What is the lysis buffer recipe and can the DNA from this protocol be used for PCR with the precise result?
Great video, you may use guanidine thiocyanate is used for RNA isolation and can solubilize proteins.
Onur Kerem Polat. You are my good friend and I am always respect this friendship.haha
hey can you please recommend me 10 books on this, im doing practical and google is not helpong
Why does the DNA sticks to the side wall of eppendorf tube at the last step of centrifugation ?
Because the eppendorf while centrifugation won't reach an horizontal state. The angle is locked so that it doesn't reach that state.
Meanwhile the acceleration vector is horizontal(because of rotation) , therefore your DNA will stack up on the side of your tube. This way it will tend to "glue" to the tube once precipitated instead of flowing with the sumernatant while you eliminate it.
Sorry for bad English.
@@ronaldmacdonald1268 Thank you so much. And yes the English is comprehensible. No sorry please.
What is the temperature for centrifugation?
Is it a technique for any microorganism's DNA isolation???
Thank you for this video, it is so nice. But I confuse is this genomic DNA or plasmid isolation and how to make lysis buffer?
genomic DNA
What is the purpose of adding chloroform into the last step?
I've tried to extract s.aureus dna from wound samples for more than 3 times but my test is negative😢 whats the reason behind failure
what method are you using for s.aureus , please tell me or give me alink . i wiill be grateful
This is because gram +ve bacteria have thick cell walls and hence are more difficult to deal with as compared to gram -ve bacteria. I am dealing with the very same problem.
One of the best tutorial.thanks
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People still do this?
what is the function of Proteinase K in this experiment?
ikmal alif this is a protease which will denature protein so that you do not get any in your lysate. Likewise, RNAse will degrade RNA.
اي بعدين هسة شتردون ..مو اضافات طلاسم ..يليتكم طقعتو بس
can we change the ratio of PCI????
Great video... It helps me a lot
Wow, that was very tedious
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Good job!
Thank you 🙏
Thanks a lot.
Very good, deep thanks
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I want to doing this experience
Muchas gracias desde chile
Excellent,thanks a mill
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Sry..Its really amazing
This is cool!!!!!
Very poor
I have question.
As we generally know some reagents sometimes nin reactibity so how could you decide that your performance is correct?