Re: [問題] DNA ladder實驗
※ 引述《by.bbs@bbs.badcow.com.tw (十年後人會在哪裡??)》之銘言：
: 1.經驗上跟cell type有很大的關係..有些cell跑出來的pattern很容易是smear-like,
: 不過這在發表時是可以被接受的.
: 2.你有測純度..那應該也同時有測濃度吧..所以你的loading量是多少呢??
: or more information about your running condition..?
: 3.H2O2的保存是老問題..它常常因保存不好而不work..有確定H2O2是ok的嗎??
: or 能造成DNA ladder的試劑很多,是否可能用其他reagent來induce?
謝謝您的指教，後來我直接寄信問廠商了，
對方說Tri-reagent不適使用於抽取小片段的DNA，
因為小片段DNA會在加了BCP離心分層時，跑到RNA（aqueous phase）那層。
我把往來信件貼在這裡給大家參考：
To whom it may concern,
My lab bought TRI reagent from your company. We isolated RNA of good
quality. And currently I also want to isolate DNA by TRI reagent and I did try
the method provided in the protocol to see if DNA laddering is generated. But
only two bands of molecular weight more than 1200bp were observed.
I want to ask, by using this method, if total genomic DNA would be
isolated? Are smaller DNA fragments also isolated? And is there any published
paper using this method to see DNA laddering? I cannot find any, would you
please send me some references? Thank you very much!
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Hello, XX,
Thank you for your email relating to the recovery of DNA from TRI Reagent
extracts. When TRI Reagent is used to extract nucleic acids from tissue samples
total genomic DNA is recovered. Some of this DNA is full length 100 kb DNA
while the remainder will be DNA that has been sheared into smaller fragments
during tissue homogenization (e.g. 20-50 kb fragments). If the tissue is
undergoing apoptosis, these smaller DNA fragments may also be distributed in
the RNA fraction. As the DNA molecule decreases in size it becomes increasingly
difficult for the reagent to distinguish between RNA and DNA simply on the
basis on the chemistry of the two molecules.
After genomic DNA is recovered from TRI Reagent homogenates and cut with
appropriate restriction enzymes it can be used for DNA profiling experiments.
In order to limit DNA shearing, laboratories that are studying restriction
length polymorphism use very gentile enzymatic methods to recovery DNA from
their samples. This minimizes shearing artifacts that are typically produced
when tissue is homogenized in TRI Reagent. However, these milder techniques
would be inappropriate for RNA isolation and this may be why you have not
seen DNA recovered with TRI Reagent or TRIzol used in this application?
As you know, the “DNA ladders” that are used to quantify PCR products,
etc. are prepared commercially and they are not derived from biological
material.
Thank you for your interest in our products. Please contact me if you have
any additional questions relating to this matter.
Sincerely yours,
Bill Wilfinger, Ph.D.
Senior Scientist
Product Development and Technical Services
Molecular Research Center, Inc.
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