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Rna Extraction

This document provides instructions for extracting and purifying total RNA from bacterial cells using an RNeasy Mini Kit. It involves lysing the bacteria, adding reagents to protect RNA, centrifuging, transferring lysate to a spin column for purification, washing with buffers, and eluting purified RNA. An optional step is described to further remove DNA contamination from the purified RNA using DNase I treatment and phenol/chloroform extraction before precipitation and resuspension of pure RNA.

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103 views3 pages

Rna Extraction

This document provides instructions for extracting and purifying total RNA from bacterial cells using an RNeasy Mini Kit. It involves lysing the bacteria, adding reagents to protect RNA, centrifuging, transferring lysate to a spin column for purification, washing with buffers, and eluting purified RNA. An optional step is described to further remove DNA contamination from the purified RNA using DNase I treatment and phenol/chloroform extraction before precipitation and resuspension of pure RNA.

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hsaddy_unej
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© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Total RNA Extraction and Purification (KIT)

A. Enzymatic Lysis of Bacteria Things to do before starting: If using RNeasy Kits for RNA purification, add 10 l -mercaptoethanol per 1 ml buffer RLT, and mix. Buffer RLT is stable for 1 month after adition of -mercaptoethanol. So Prepare on small tube (in eppendorf tube). A1. Prepare TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) containing 10 mg/ml lysozyme. Prepare in eppendorf tube for 1 ml A2. Calculate the required volume of bacterial culture (use 3 ml) A3. Pipet 2 volume of RNAprotect Bacteria Reagent into a tube Pipet 6 ml of RNAprotect Bacteria Reagent into a labcon tube (max volume is 15 ml). A4. Add 1 volume of bacterial culture to the tube. Mix immediately by vortexing for 5 s. Incubate for 5 minutes at room temperature (15-25OC). Add 3 ml of bacterial culture to the tube which have added with 6 ml of RNAprotect Bacteria reagent. A5. Centrifuge for 10 minutes at 5000 x g Centrifuge for 10 minutes at 6.20 rpm using rotor No. 25 on HIMAC CR21E centrifuge at 20OC. A6. Decant the supernatant. Remove residual supernatant by gently dabbing the inverted tube once onto a paper tower. After dabbing, do not remove any remaing supernatant by pipetting. A7. Add the appropriate volume of TE buffer containing lysozyme Add 100 l TE buffer containing lysozyme. A8. Mix by vortexing for 10 s. Incubate at room temperature (15-25OC) for 10 minutes. During incubation, vortex for 10 s at least every 2 minutes. A9. Add the appropriate volume of Buffer RLT and vortex vigorously. If particular material is visible, pellet it by centrifugation, and use only the supernatant in step A10. Add 350 l of buffer RLT and vortex. Transfer the mixture to the new eppendorf tube. Then centrifuge for 2 minute using microfuge, and transfer 450 l supernatant to new eppendorf tube. A10. Add appropriate volume of ethanol (96-100%). Mix by pipetting (RNeasy Mini procedure). Add 250 l of ethanol 99.5%, mix and keep the mixture to next step (Purification).

B. Purification of Total RNA from bacteria Using the RNeasy Mini Kit. Things to do before starting: Buffer RPE is supplied as a concentrated. Before using for the first time, add 4 volume of ethanol (99.5%) to obtain working solution. For example 250 ul of RPE concentrated buffer with 750 ul of 99.5% ethanol (Use eppendorf tube and make it onto 2 tubes). B1. Transfer up to 700 ul lysate, including any precipitate tha my have formed, to an RNeasy Mini spin column placed in a 2 ml collection tube (supplied). Close lid gently, and centrifuge for 15 s at 8000 x g (>10.000 rpm). Discard the flow-through. Reuse the collection tube in the step B2. Add 700 ul of lysate from Enzymatic Lysis procedures to RNeasy Mini spin tube set. Microfuge for 15 s. Discard flow-through. B2. Add 700 ul buffer RW1 to the RNeasy Mini spin column. Close lid gently, and centrifuge for 15 s at 8000 x g (>10.000 rpm) to wash the spin column membrane. Discard the flow-through and collection tube. B3. Place the RNeasy Mini spin column in a new 2 ml collection tube (supplied). Add 500 ul Buffer RPE to the RNeasy Mini spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g (>10.000 rpm) to wash spin column membrane. Discard the flow-through. Reuse the collection tube in step B4. B4. Add 500 ul Buffer RPE to the RNeasy Mini spin column. Close the lid gently, and centrifuge for 2 minutes at 8000 x g (>10.000 rpm) to wash spin column membrane. Do not touch the flow-through with spin column. B5. Place the RNeasy Mini spin colum in a new 1.5 ml collection tube (supplied). Add 30-50 ul Rnase-free water directly to the spin column membrane. Close the lid gently, an centrifuge for 1 minute at 8000 x g (>10.000 rpm) to elute the RNA. B6. If the expected RNA yield is > 3 ug, repeat step B5 using another 30-50 ul of RNase-free water directly to the spin column membrane. Close the lid gently, an centrifuge for 1 minute at 8000 x g (>10.000 rpm) to elute the RNA. Reuse the collection tube from step B5.

REMOVING DNA Contaminant on Total RNA


Extraction and purification of Total RNA from bacterial cell is not absolutely free contaminant of DNA. So, after extraction and purification Total RNA (Usually using Kit), Removing of DNA contaminant is needed to get very pure Total RNA which need in gene expression such as RT-PCR or/and Quantitative-PCR. After getting Crude total RNA using kit (RNeasy mini spin column), next step is needed. 1. Prepare the following reaction mixture. a. Total RNA (20~50 ug) : 30 ul b. 10 X Dnase I buffer : 5 ul c. Recombinant Dnase I (Rnase-free) (10 U) : 2 ul d. Ribonuclease inhibitor (20 U) : 4 ul e. DEPC-treated water (up to 50 ul of total volume) : 9 ul 2. Incubate for 20~30 minutes (Take 30 minutes). 3. Perform Phenol/Chloroform extraction a. Mix 50 ul of DEPC-treated water b. Add 100 ul of PCI (Phenol/Chloroform/Isoamylalcohol) (25:49:1) c. Centrifuge at 12.000 rpm for 5 minutes at room temperature d. Transfer the upper layer to a new tube (95 ul) e. Add equal amount (95 ul) of Chloroform/Isoamylalcohol (24:1) f. Mix by inverting tube carefuly. g. Centrifuge at 12.000 rpm for 5 minute at room temperature h. Transfer the upper layer to a new tube (95 ul). 4. Add 10 ul of 3M sodium acetate and mix slowly 5. Add 250 ul of Chilled 99.5% ethanol and mix 6. Keep on -80OC for 20 minutes. 7. Centrifuge at 12.000 rpm for 10 minute at 4OC, 8. Remove supernatant. 9. Wash the precipitate with 200 ul of chilled 70% ethanol. 10. Centrifuge at 12.000 rpm for 5 minute at 4OC, 11. Remove supernatant and dry precipitate 12. Dissolve precipitate in a suitable amount of DEPC-treated water (30 ul). 13. Check the presence of DNA contaminant.

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