互联网2013-11-13
1. 2-mercaptoethanol
2. Absolute (96%-100%) ethanol
3. Liquid nitrogen for freezing/disrupting samples
1. Microcentrifuge capable of 10,000 x g
2. Nuclease-free microfuge tubes
3. Preheat an aliquot (100 ul per sample) of DEPC-treated water at 65°C
1. Freeze and grind up to 100 mg arthropod tissue under liquid nitrogen. Grind tissue completely to obtain a fine homogenous powder.
2. Immediately add 500ul Buffer RB/2-mercaptoethanol. Add 20ul 2-mercaptoethanol per 1ml of Buffer RB and then add 500ul of this mixture to the sample. Samples should not be allowed to thaw before Buffer RB/2-mercaptoethanol is added. Vortex vigorously to make sure that all clumps are dispersed. RNA cannot be effectively extracted from clumped tissue.
Note: Add 20ul 2-mercaptoethanol per 1 ml of Buffer RB before use. This mixture can be made and stored at room temperature for 1 week.
3. Pipet the lysate directly into a Homogenization Spin Column placed in 2ml collection tube. Centrifuge at .13,000 x g for 5 min at room temperature.
4. Carefully transfer the supernatant of the flow-through fraction to a new 1.5ml microfuge tube, making sure not to disturb the pellet or transfer any debris. Add 0.5 volume absolute ethanol and mix by vortexing at maxi speed for 15 seconds. If anyprecipitation can be seen at this point, break the precipitation by passing through a needle using a syringe or pipetting up and down 10-15 times.
TIP: In most cases 450 ul supernatant can easily be removed. This will require 225ul ethanol. Note that depending on the sample, the volume of supernatant may vary. After transferring to a fresh tube, measure the volume and add the correct amount of ethanol.
5. Apply the entire sample, including any precipitates that may form to a HiBind® RNA Mini column assembled in a clean 2 ml collecting tube (supplied). Close the cap gently. Centrifuge at 10,000 x g for 30 sec at room temperature. Discard the flow-through liquid and place the column back into the collecting tube.
Optional on-membrane DNase I digestion: This is the starting point to perform DNase I digestion. See Page 8 for detailed protocol.
6. Add 500 ul RNA Wash Buffer I, close the tube gently. Centrifuge at 10,000 x g for 30 sec. Discard both flow-through liquid and collecting tube.
7. Place column in a clean 2 ml collection tube (supplied), and add 700 ul RNA Wash Buffer II diluted with ethanol. Close the column gently, Centrifuge at 10,000 x g for 30 sec at room temperature and discard flowthrough. Re-use the collection tube in step 7.
Note: RNA Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.
8. Wash column with a second 500 ul of RNA Wash Buffer II by repeating step 6. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin cartridge for 1 min at full speed to completely dry the HiBind® matrix.
9. Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and elute the RNA with 50-100 ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 1 min at maximum speed. A second elution into the same tube may be necessary if the expected yield of RNA >50 ug.
Note: RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution.
No RNA extraction procedure can completely remove genomic DNA. For sensitive work (such as RT-PCR or differential display) we suggest that you treat the eluted RNA with RNase-free DNase. Also for RT-PCR, use intron-spanning primers that allow easy identification of DNA-contamination. A control PCR reaction containing the RNA as template will also allow detection of DNA contamination. For designing intronspanning primers, call our technical staff at 800-832-8896 for assistance. We can help design primers suited to your needs.
相关产品推荐
mRNA纯化试剂盒|高效、灵敏、高性价比| mRNA Isolation Master Kit
￥2600
全能型植物RNA提取试剂盒 (DNase I)，阿拉丁
￥1197.90
ReadiPrep™ Lipopolysaccharide (LPS) Isolation Kit
￥66
ReadiPrep™ Exosome Isolation Kit
ExoQuick-CG cGMP级外泌体分离试剂（50 mL）-ExoQuick-CG cGMP-grade Exosome Isolation reagent (50 mL)
￥55
相关问答
问
RIP出的RNA，怎么去检测具体有哪些RNA呢
RNA干扰提取RNA注意事项？
怎么用T4 RNA 连接酶链接RNA分子呢？
相关方法
RNA 提取
2023-04-09
RNA印迹
2022-02-11
分离RNA
2024-05-14
推荐阅读
Isolation of Zebrafish Gonads for RNA Isolation
RNA提取--RNA ISOLATION
Isolation of DNA and RNA
