phenol-chloroform-extraction - (Sep/26/2005 )
I have a question as well, what happens if you skip the phenol extraction, let's say before using a restriction enzyme? Is it ok if I do it after the reaction with the restriction enzyme?
well if you forget to do this before, you may see degradation of your DNA. You'll see in agarose gel.
Most important is that the restriction enzyme will not be efficient.
So it's ok in the sense that you can do phenol extraction after restriction and redo the restriction.
dear friend:
i read ur suggestion about prepare phenol solution from crystal form,
in case of RNA extraction & THE QUESTION IS if this preparation give me saturated phenol or not
& if it is important to use saturated phenol in the extraction & by the same way what is the differance between saturated & unsaturated phenol.
i hope 2 help me in this topic thx u very much regards.
IN PHENOL-CHLOROFORM DNA EXTRACTION WE USE PHENOL,BUT IN LIQUID CONDITION.
I ORDERED PHENOL BUT THE ARRIVED ONE WAS IN CRYSTAL CONDITION (MERCK).
I CANT FIND ANY INFO ABOUT THAT.
HOW SHOULD I PREPARE LIQUID-PHENOL FROM CRYSTAL FORM.
THX.
you can dissolve 500g crystal phenol in 500 ml 1M tris pH 8.0 (if you want phenol solution for DNA extraction) or 50 mM Sodium acetate pH 4.0 (when prepare acid Phenol for RNA extraction). Keep in mind check pH of phenol solution before use and discard this solution when phenol turns red/brown.
Hey,
I also wrote this in a separate topic but... yeah, I think I can detail the question. I have been doing Ph-Ch extraction from around 10^8 cells ,Gram (-), with Tri reagent to denature -self made, working perfectly- my student was also trying the same protocol and he said he did not get any RNA one day.. then I repeated the whole thing several times it was true that all of a sudden my protocol started to yield no RNA what so ever -I mean, I measure something with Nanodrop but you don't see anything on the gel- It is exactly same culture, same solutions, same tubes and same protocol -which is normally very easy going- and all of a sudden it starts not to yield any RNA....
any suggestion?
hai...
just some info to tell. isoamylalcohol addition in extraction protocol also act to stabilize formation of both phase after we centrifuged it.
[font="Comic Sans MS"][/font]hi.....
im a new member of this forum.....
im working on dna extraction from hair follicles....
initially im facing problems in getting the dna in appreciable amounts. can sm 1 tell me the stepwise procedure from cell lysis to its purification......?
thnx
I have the same problem with her...any suggestion ???
Thank you
i hv tried this but still the DNA sample is very viscous and difficult to load.
waiting for ur reply
satyam
from ambion's website :
Efficient extraction of cell extracts or solutions containing nucleic acid are most often performed with a series of phenol and phenol:chloroform extractions at a specific pH. Both phenol and chloroform cause proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase. After centrifugation, the aqueous phase containing nucleic acid is re-extracted with an equal volume of chloroform:isoamyl alcohol (1,3,4). This combination of extractions is thought to reduce the loss of RNA due to the formation of insoluble protein:RNA complexes at the interphase.
Chloroform is mixed with phenol to increase the efficiency of nucleic acid extractions by reducing losses of RNA at the interphase. The increased efficiency is due to chloroform's ability to denature proteins and aid in the removal of lipids, thus improving separation of nucleic acid into the aqueous phase. Phase separation is also enhanced, which assists in the removal of the aqueous phase with minimal cross contamination from the organic phase. Often isoamyl alcohol is added to phenol:chloroform to reduce foaming
fred