[求救] in vitro transcription 做不出來
因為實驗的需要,我是用RNAII promotor 的PCR片段當template,promotor下游還有接
100bp序列。標定的系統是用roche-DIG,in vitro transcription完後做northern,沒
有hybridization 直接壓片。
5x transcription buffer (paper上寫的)
STOCK WORK
Tris-Hcl 0.2M 40
KCl 0.75M 10
MgCl 50mM 160
glycerol 5% 1%
DTT 2mM 0.4
----------------------------------------------
reaction
template(60.4ng/ul) 3ul
5xtranscription buffer 4ul
DIG-NTP MIX(10mM) 2ul
RNA polymerase(0.7ug/ul) 2ul
RNAse inhibitor(40U/ul) 2ul
DEPC-H2O 7.3ul
↓37度,兩小時
加入 2ul 0.2M EDTA 終止反應
20ul loading dye
loading dye(roche的配方)
250 ul of 100% Formamide
83ul of 37% Formaldehyde
50 ul 10 × MOPS
50 ul 100% Glycerol
10 ul 2.5% Bromphenolblue
57 ul DEPC/DMPC treated water
↓65度,10min
↓冰上 2min
↓run 1.5% Formaldehyd
2.25 g Agarose
141.9 ml 1 × MOPS
8.1 ml 37% Formaldehyde
running buffer:1X MOPS
↓50V,兩個小時
transfer overnight→detect →壓片
---------------------------------------------
我在跑膠的時候有loading control RNA,壓片有壓出來,可是我的sample一片空白,
表示我跑膠之後的方法是沒錯的,所以是出在我的sample根本沒有轉出RNA吧!?
麻煩大家幫我看看是怎麼回事啊~~~~~~~~
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※ 編輯: sunday30430 來自: 163.25.118.160 (07/27 18:56)
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