我要登录|
免费注册
|
我的丁香通
- 企业机构：
- 成为企业机构
- 个人用户：
- 个人中心
移动端

大家都在搜

大家都在搜

0 人通过求购买到了急需的产品

免费发布求购

点赞

收藏

分享

Modified Total RNA Protocol for Heart, Muscle, Brain and Skin tissue

互联网2013-11-13

684

实验步骤

Note: Always use less starting material (<500mg) for the first time isolation to ensure a good result. If results obtained are satisfactory increase amount of starting material.

1. Disrupt the tissue and homogenize lysate in 6.0mL TRK lysis buffer with a rotorstator homogenizer. Transfer the homogenate to a 50ml centrifuge tube which is capable of 3500 x g.

2. Add 15ml ddH₂ O to the homogenate and add 220 ul Proteinase K or OB Protease (20mg/ml). Vortex for 30 seconds to mix. Incubate at 60°C for 15- 30 minutes.

3. Centrifuge at 3500 x g for 10 minutes and transfer clear supernatant (around 22.0ml) to a new 50 ml tube. (Avoid any precipitate or film on top of the supernatant may formed during process of step 2.)

4. Add 0.5 volume of absolute ethanol (11ml) to the sample and mix with pipetting.

5. Transfer 13ml of the mixture including any precipitate may have formed in step 4 to the HiBind^® RNA Maxi-column placed in a 50ml centrifuge tube (supplied). Closed the tube and centrifuge at 3500-5000 x g for 10 minutes. Discard the flow-through. Re-use the tube for next step.

6. Repeat step 5 twice until all the mixture from step 4 pass through the HiBind^® RNA Maxi-column.

7. Add 15 ml RNA Wash Buffer I, cap the 50 ml collecting tube and centrifuge for 5 min at 3,500-5,000 x g at room temperature. Discard flow-through liquid and reuse 50 ml tube in step 8.

8. Add 15 ml RNA Wash Buffer II, cap the 50 ml collecting tube and centrifuge for 5 min at 3,500-5,000 x g at room temperature. Again, discard flow-through liquid and reuse 50 ml tube in step 9.

9. Repeat step 8 with another 15 ml RNA Wash Buffer II. Discard flow-through liquid and re-insert the spin column in the empty 50 ml collecting tube. Centrifuge the empty column for 10 min at 5,000 x g to dry the HiBind^® matrix. This step is critical for removing traces of ethanol that may otherwise interfere with subsequent downstream applications.

Note: When a vacuum oven is available, place the maxi column into a vacuum oven which is preset at 60°C for 10-15 minutes. This will ensure that the column can be completely dried before elution.

10. RNA elution: transfer the HiBind^® RNA maxi column to a new 50 ml centrifuge tube (not supplied) and pipet 850 ul DEPC-treated water directly onto the matrix. For expected RNA yields greater than 1 mg use 1.25 ml of water. Cap the tube and allow the matrix to soak for 1 min. Centrifuge for 5 min at 5,000 x g.

Tip: Yield will be increased by 30%-70% by repeating the elution with a second volume of DEPC-treated water. For a higher final RNA concentration, this second elution may be performed using the first eluate. However, this will result in a ~30% lower overall yield.

ad image

ad image

相关产品推荐

链特异性RNA建库试剂SMART-Seq® Total RNA Pico Input with UMIs (ZapR™ Mammalian)

￥18658

Coagulation Factor III / Tissue Factor / CD142 鼠单抗 (FITC)

￥700

Creatine Kinase Muscle/CKM 兔多抗（抗原亲和纯化）

￥1699

Dulbecco 磷酸盐缓冲盐水，用于细胞培养, dry powder, DPBS Modified, without calcium, without magnesium，阿拉丁

￥197.90

GJA1/GJA1蛋白Recombinant Human Gap junction alpha-1 protein (GJA1)重组蛋白Connexin-43 Short name: Cx43 Gap junction 43KDA heart protein蛋白

￥1344

相关问答

问

求一份详细的ABE protocol

问

抗体包被的protocol

问

转投American Heart Journal PLUS

3 回答 674 围观

相关方法

Strep 标签蛋白纯化 protocol

2023-06-02

植物单细胞测序 protocol——水稻花序原生质体制备方法

2023-06-01

RNA 提取

2023-04-09

推荐阅读

Total RNA isolation protocol

心肌 heart muscle， cardiac muscle

RNASafer® Protocol for Total RNA Isolation

关于丁香通

公司信息

个人用户

企业机构

无忧采购轻松科研

无忧采购轻松科研

提问

扫一扫

丁香实验小程序二维码

实验小助手

丁香实验公众号二维码

扫码领资料

反馈

TOP

打开小程序