I am currently looking into the extraction of DNA from E. coli.
I want to extract the DNA using a manual method and also using centrifuge.
Once I have extracted the DNA I want to test it for yield, purity and possibly run gel electrophoresis to determine molecular weight.
I have found some protocols online but I am stuck on a few things. Uses the centrifuge method a couple of this are not well explained.
They use a Lysis solution which is SDS and also use RNase solution. But the protocol also uses a Protein Precipitation Solution (PPS) and a DNA Rehydration Solution (RH) but does not say what it is made up of.
Can anyone help clear this up for me?
As once the DNA has been extracted I want to test for purity in the spectrophotometer.
Thanks
Rezarf
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6 replies to this topic
#2
Adrian K
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Posted 20 March 2011 - 07:40 AM
Actually, if you just want to PCR to clone a gene, a tip-touch method is more than sufficient: use a tip, touch a colony and suspend into PCR tube with primers and master mix.
Protein precipitate solutions usually is ammonium acetate and DNA Rehydration solution usually is TE buffer.
Protein precipitate solutions usually is ammonium acetate and DNA Rehydration solution usually is TE buffer.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#3
Rezarf
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Posted 20 March 2011 - 11:44 AM
adrian kohsf, on 20 March 2011 - 07:40 AM, said:
Actually, if you just want to PCR to clone a gene, a tip-touch method is more than sufficient: use a tip, touch a colony and suspend into PCR tube with primers and master mix.
Protein precipitate solutions usually is ammonium acetate and DNA Rehydration solution usually is TE buffer.
Protein precipitate solutions usually is ammonium acetate and DNA Rehydration solution usually is TE buffer.
hi thanks for the reply,
I would like to use the PCR but I have been told by my supervisor that we cant use the PCR machine due to money issues LOL. So got to do it the long way.
Thanks for the info on the ammonium acetate.
Rezarf
#4
Adrian K
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Posted 21 March 2011 - 05:25 PM
Welcome, and all the best in your work.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#5
merlav
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Posted 25 March 2011 - 07:43 AM
Also for protein precipitation you can use sodium chloride at very high concentration ( for prepare just added NACl until no more in solution). When doing the extraction at the step of PP incubate on ice for 5 min before centrifuge.
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I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
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Albert Einstein
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie
#6
Rezarf
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Posted 28 March 2011 - 04:26 AM
The lab tech says they do not have Ammonium Acetate nor does it have phenol:chloroform so looks like it will be sodium chloride. Should I add a protease to break down the proteins?
So once i add the PP do i then incubate on ice for 5 mins before centrifuge?
Thanks
Rezarf
So once i add the PP do i then incubate on ice for 5 mins before centrifuge?
Thanks
Rezarf
#7
Adrian K
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Posted 28 March 2011 - 09:25 PM
I not sure about Sodium chloride. Do you have Sodium acetate or any "*acetate"? perhaps it might be able to substitute...
Can you refer me to the link?
Yes, "usually" you have to incubate on ice before centrifuge for that step, if I remember correctly.
Can you refer me to the link?
Yes, "usually" you have to incubate on ice before centrifuge for that step, if I remember correctly.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
