上欣 asked in 科學生物學 · 1 decade ago

DNA hybridization procedure !

Hi :

I want to ask some question about single strand DNA (ssDNA) hybridization !

I am using three artifical ssDNA with complementary sequence

oligo 1. 5'-TCTCAACTCGTA-3'

oligo 2. 5'-TACGAGTTGAGAATCCTGAATGCG-3'

oligo 3. 5'-CGCATTCAGGAT-3'

My questions are :

1. when we got the sample (powder form) from vander, what kind of solutions we need to use to make DNA solution?

Some article suggest TE-buffer with pH=7.4

2. when we dissolve ssDNA in this solution, low temperature environment is required ?

some article suggest 4C, 3hr shaking

3. the NaCl concentration for ssDNA hybridization and temperature and time ?

some article suggest 0.1M NaCl, T<Tm, time > 3hr

It will be a great help if anyone can give me a detail prodecure for this !!

Thank you and have a good day.

PS, I can read chinese but I can't type it in my computer, sorry about that

Update:

by the way,

here is the web site I use to get a roughly idea about DNA hybridization condition

http://www.idtdna.com/analyzer/Applications/OligoA...

if you type your DNA sequence, you can get roughly Tm, or it will form hairpin loop or not.

1 Answer

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  • 1 decade ago
    Favorite Answer

    1.溶解ssDNA用TE buffer是一般比較常見的,只要不是要接著做restriction enzyme digestion都很合適

    至於TE buffer的濃度與pH值,一般而言用一倍TE即可(10mM Tris-HCl, and 1mM EDTA) pH值的範圍在7.4~8.0之間都是通用的

    2.溶解時的溫度其實並沒有太大的差異

    如果趕著作實驗,RT數十分鐘加輕微的拍打(不建議用vortex)即可

    不趕時間的話4℃或置冰上,一至三小時或更久也無妨(當然不建議overnight)

    如果時間很趕,37℃五分鐘加拍打也可以

    因為TE本身就會對DNA有保護的功能,所以溫度時間都還不會有太大影響,只要不要太低溫或太高溫即可

    3.Hybridization的condition則需要看你的實驗來決定,是Southern blotting或是in situ hybridization或是colony hybridization實驗不同condition也會不同。

    像我自己最近在作DIG的Southern,它的條件就是

    0.75M NaCl

    75mM Sodium Citrate

    0.02% SDS

    1% Blocking reagent

    0.1% N-Lauroylsarcosine

    又因為我做的是single copy的genomic DNA Southern blot,所以是42℃ 16hr,不過我本身的probe是dsDNA

    所以這可能就要看你的實驗設計與需要了

    個人慘痛的建議是,可以先做做看,如果一次成功當然是最好,如果失敗了,從失敗中找尋修正的方法也是不錯啊...(淚奔)

    以上 希望對你有幫助

    Source(s): 自己實驗的經驗,Roche DIG Application Manual
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