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

E.Z.N.A.® Protocol for Bacteria

互联网2013-11-13

1319

实验试剂

Additional materials to be supplied by user

1. RNase-free Lysozyme

2. TE buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA) Procedure

实验步骤

1. Harvest Cells and resuspend in 500 ul TE/lysozyme and incubate at RT for 7 min. Centrifuge 1010 cells at 4,000 x g for 5 min. Discard supernatant and add 100 ul of TE buffer containing lysozyme (0.5 mg/ml for Gram-negative and 4 mg/ml for Gram-positive bacteria). Resuspend cells completely and incubate at room temperature for 7 min.

2. Add 2 ml of TRK Lysis Buffer and mix by pipetting several times. Remember to add 20 ul of ß-mercaptoethanol per 1 ml of TRK Lysis Buffer.

3. Add 1.5 ml 100% ethanol to lysate and mix by vortexing. A precipitate may form at this point. This will not interfere with RNA purification.

4. Apply sample (approximately 3.5 ml) from step 3 to an HiBind^® RNA Midispin column. With the column mounted in a clean 15 ml collection tube (supplied with kit) centrifuge 5 min at 5000 x g (at room temperature) in a centrifuge. Discard flow-through and proceed to step 5. (This is the starting point for optional DNase I digestion treatment, see page 6 for protocol)

5. Wash column with 3ml RNA Wash Buffer I. Centrifuge 5 min at maximum speed and discard both flow-through and collecting tube.

6. Place spin column into a 8 ml clean collection tube (supplied) and add 3 ml RNA Wash Buffer II diluted with ethanol. Centrifuge and discard flow-through as above. Reuse the collection tube in step 7.

Note: Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.

7. Wash column with a second 3 ml 1 X Wash Buffer II. Repeat step 6 and discard flow-through. Then empty the collection tube and centrifuge the spin cartridge for 10 min at 4000-5000 x g to completely dry the HiBind™ matrix.

8. RNA Elution. Transfer column to a clean 15 ml collection tube (Not supplied) and elute RNA with 250-500ul DEPC-treated water (supplied with kit). Centrifuge column for 10 min at 4,000-5,000 x g. If the expected RNA yield > 500 ug the a second elution may be required. Elution with two 250 ul aliquots is no more efficient than with one 500 ul aliquot.

注意事项

All centrifugation steps must be carried out at room temperature.

ad image

ad image

相关产品推荐

单宁酸溶液;1401-55-4;25 grams + 100 mL Waterfor Direct Count for Bacteria, Yeast, and Mold;V32785-50ml

￥205

Zymo-Spin™ ChIP Kit (25 preps) Mechanical Shearing Protocol ~undefined'

￥3031

E.Z.N.A.™ BAC/PAC DNA Kit

￥550

pLV3-CMV-Mem-cbpD(bacteria)-6×His-EF1a-CopGFP-Puro质粒载体

￥680

细菌DNA提取试剂盒 FastPure Bacteria DNA Isolation Mini Kit（DC103）

￥665

相关问答

问

大家是怎么溶解蕨素Z的呀

问

大家是怎么溶解蕨素Z的呀

问

普利莱E1015

4 回答 444 围观

相关方法

用SGM Plus®进行扩增

2024-05-14

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

推荐阅读

E.Z.N.A.® Viral RNA Spin Protocol

E-Z 96® Viral RNA Protocol with Centrifugation

E.Z.N.A.® Mag-Bind® Viral DNARNA Spin Protocol

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