Mesoplasma florum:RNA Purification

RNeasy Protect Bacteria Mini Kit (Qiagen Cat. No. 74524)

Before starting add 10 ul β-mercaptoethanol per 1 ml Buffer RLT and 4 volumes of 96-100% ethanol to Buffer RPE

Cell Lysis

• Start with 1 to 2 ml liquid culture.
• Add 2 volumes RNAprotect Bacteria Reagent. Mix immediately by vortexing for 5 seconds.
• Incubate at room temperature for 5 minutes.
• Centrifuge for 10 minutes at 5000g.
  • There may not be a visible pellet after centrifugation, which is not a problem.
• Pour out the supernatant and remove any residual liquid by gently dabbing the inverted tube once onto a paper towel.
• Add 10 ul Proteinase K and 200 ul TE to the pellet. Carefully resuspend by pipetting up and down. Vortex for 10 seconds to mix.
• Incubate at room temperature for 10 minutes. Use either a shaker-incubator or vortex for 10 seconds at least every two minutes.
• Add 700 ul Buffer RLT and vortex. If particulate material is visible, pellet it and use only the supernatant
• Add 500 ul Ethanol (96-100%) and mix by pipetting.

RNA Purification

• Transfer 700 ul lysate to a spin column placed in a 2 ml collection tube. Close the lid gently and centrifuge for 15 seconds at ≥8000g (≥10,000 rpm). Discard the flow-through (do not mix FT with bleach). Repeat until all the lysate has been centrifuged through the column.
• Add 350 ul Buffer RW1 to the column and centrifuge for 15 seconds at ≥8000g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through (do not mix the FT with bleach).
• Add 10 ul DNase I stock solution to 70ul Buffer RDD. Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube. DO NOT vortex, it will denature DNase I.
• Add the DNase I mix directly to the column membrane and incubate at room temperature for 15 minutes.
• Add 350 ul Buffer RW1 To the column, wait for 5 minutes, and then centrifuge for 15 seconds at ≥8000g (≥10,000 rpm). Discard the flow-through.
• Place the column in a new 2 ml collection tube. Add 500 ul Buffer RPE to the column, close the lid gently, and centrifuge for 15 seconds at ≥8000g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
• Add 500 ul Buffer RPE to the column, close the lid, and centrifuge for 2 minutes at ≥8000g (≥10,000 rpm).
• Place the column in a new 1.5 ml collection tube. Add 30-50 ul RNase-free water directly to the column membrane. Close the lid and centrifuge for 1 minute at ≥8000g (≥10,000 rpm) to elute the RNA.
• If the expected yield is >30 ug, repeat the previous step using another 30-50 ul of RNase-free water, or the water that was already used to elute.

Preparing DNase I (From Qiagen Cat. No. 79254)

• Dissolve the solid DNase I in 550 ul RNase-free water. Inject the water into the vial using a needle and syringe.
• Mix gently by inverting the vial. DO NOT vortex.
• For long-term storage, remove the solution from the vial, divide into single-use aliquots, and store at -20°C for up to 9 months. Thawed aliquots can be stored at 4°C for up to 6 weeks. Do not refreeze aliquots once thawed.

Notes 7/1/08 TK

• Started with 600 ul in 2 ml tube, add 1200 ul RNA Protect
• Vortex and incubate 10 minutes at RT
• Spin down 5 min at 17000g
• Decant supernatent, blot tube mouth dry
• Add 200 ul TE
• Add 10 ul proteinase-K, vortex
• incubate 10 min at RT
  • solution cloudy
• Add 700 ul RLT
• Add 500 ul EtOH
  • Some genomic DNA visible in solution, solution clears
• Spin twice through a column with 700 ul each
• Add 700 ul RW1, sit for 5 minutes, spin through
• Switch column to a new tube
• Wash 3x with 500 ul RPE
• Spin dry 1 min at 17000g
• Transfer to a 1.5 ml tube
• Elute 2x with 40 ul RNAse free water
• Run 1 ul and 3 ul on an E-Gel
• Measure on nanodrop: 223 ng/ul in 70 ul, ratio 2.1
• Gel shows clear 16S and 23S bands