RNA extraction

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(Prepare bench)
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#Wipe down thoroughly with EtOH
#Wipe down thoroughly with EtOH
#Use Rnase Zap wipes to clean bench and pipettes  
#Use Rnase Zap wipes to clean bench and pipettes  
-
#Wipe all down with paper towel/ H2O, 2x.
+
#Wipe all down with a wet (H20) paper towel (2x).
==Culture growth and sample collection==
==Culture growth and sample collection==

Revision as of 17:15, 6 June 2005

cmc, adapted from hkeller

Contents

Prepare RNase-Diminished bench

  1. Wipe down thoroughly with EtOH
  2. Use Rnase Zap wipes to clean bench and pipettes
  3. Wipe all down with a wet (H20) paper towel (2x).

Culture growth and sample collection

  1. Samples from continuous culture in chemostat:
    1. Chemostat SOC
    2. At desired time, measure OD600 of chemostat culture
    3. Pull 2mL sample from chemostat through bubbler line. Immediately proceed to cell collection.
  2. Samples from batch culture:
    1. Inoculate 5mL standard media (see protocol, M9_recipe) from fresh plate (<2 weeks). Grow to saturation at 37° C, approx 20 hr.
    2. Measure OD600 of saturated cultures. All should be ~2.5.
    3. Dilute all saturated cultures to OD600=0.0025 (dilution ~1:1000) into 5mL fresh media. (5μL culture if OD600=2.5).
    4. Grow to mid-log, OD600 ~ 0.4
    5. Pull 2mL sample and proceed immediately to cell collection.

Cell collection

  1. Immediately combine 0.8mL cold stop solution with each 1mL sample in a pre-chilled centrifuge tube. (Stop solution= 5% H2O saturated phenol in ethanol).
  2. Store on ice while plating dilutions of culture for CFU count. Plate three replicates of a 1:104 and a 1:105 dilution.
  3. Centrifuge samples at 4°C in Sauer Lab tabletop centrifuge. 13,000rpm, 10 minutes.
  4. Decant the supernatants from the pellets and discard.
  5. (optional) At this point, the pellets can be frozen at -80°C until ready to proceed with step 4. Thaw on ice before lysis.

Lysis

  1. Resuspend each pellet in 0.5 ml of Lysis buffer (2% SDS and 4 mM EDTA).
  2. Incubate the cells in a 100°C sand bath for 3 minutes to lyse the cells.
  3. Add 29.3 μl of 3M NaOAc to each tube and transfer to ice.
  4. (optional) spike in control mRNA to evaluate the efficiency of extraction

Phenol Extraction with heat

  1. Add an equal volume (0.5 mL) of water-saturated phenol to each tube.
  2. Invert several times to mix.
  3. Transfer to a 67°C water bath for 6 minutes and invert every 40 seconds.
  4. Immediately transfer to ice.
  5. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 10 minutes.
  6. Transfer as much of each aqueous layer as possible to new tubes. The aqueous layer is the upper layer and is distinct from the organic phase which is tinted yellow by 8-Quinolinol in the phenol

Phenol/Chloroform Extraction

  1. Add an equal volume (0.5 mL) of water-saturated phenol:chloroform:isolamyl alcohol (25:24:1) to each tube.
  2. Invert several times to mix.
  3. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
  4. Transfer as much of each aqueous layer as possible to new tubes.
  5. Repeat phenol/chloroform extraction once.

Ethanol Precipitation

  1. To each tube, add: 1/10 volume (50μl) 3M NaOAc, 1/10 volume (50 μl) 1 mM EDTA, and 2-2.5 volumes (1 mL) of cold, 100% ethanol.
  2. Invert to mix and then incubate at -80°C for 20 minutes.
  3. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 25 minutes.
  4. Decant the ethanol from the pellets and discard.
  5. Wash each pellet in 1 ml of cold 80% ethanol.
  6. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
  7. Decant the ethanol from the pellets and discard.
  8. Repeat wash with cold 80% EtOH twice, for a total of three washes.
  9. Dry the pellets in a 37°C heat block after the final wash is removed, ~15 min
  10. Resuspend each pellet in 50 μl of Buffer EB.
  11. (Optional) Pool replicates of each sample in order to increase the total cellular RNA per sample (helpful for Northern Blot or RPA, not necessary for RT-PCR.)

Absorbance reading

  • Nanodrop (make sure to set for RNA). Record concentration and A260/A280 ratio. An ideal purity has an A260/A280 between 1.8 and 2.2.

Storage

  • If the sample is to be treated immediately with DNase, proceed to the next section. Otherwise, dilute and/or aliquot the samples if necessary and store at

-20°C until needed.

DNase treatment

  1. Combine:
    • 50 μL of 10x DNase I Buffer
    • 50 – 100 L of RNA Extraction product
    • 2 μL of Superase Inhibitor
    • 5 μL DNase I (~1 ug enzyme)
    • H2O (RNase-free) to total volume of 500 μL
  2. Incubate 10 min at 37°C.
  3. Optional: If using NEB DNase I, heat inactivate 10 min at 75°C.

Phenol/Chloroform Extraction

  1. Add an equal volume (0.5 mL) of water-saturated phenol:chloroform:isolamyl alcohol (25:24:1) to each tube.
  2. Invert several times to mix.
  3. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
  4. Transfer as much of each aqueous layer as possible to new tubes.

Ethanol Precipitation

  1. To each tube, add: 1/10 volume (50μl) 3M NaOAc, 1/10 volume (50 μl) 1 mM EDTA, and 2-2.5 volumes (1 mL) of cold, 100% ethanol.
  2. Invert to mix and then incubate at -80°C for 20 minutes.
  3. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 25 minutes.
  4. Decant the ethanol from the pellets and discard.
  5. Wash each pellet in 1 ml of cold 80% ethanol.
  6. Centrifuge in an Eppendorf 5414 table top microcentrifuge (15,000 RPM) at 4°C for 5 minutes.
  7. Decant the ethanol from the pellets and discard.
  8. Repeat wash with cold 80% EtOH twice, for a total of three washes.
  9. Dry the pellets in a 37°C heat block after the final wash is removed, ~15 min.
  10. Resuspend each pellet in 30 μl of nuclease free water if to be used in RPA or in 10 μL Buffer EB if to be used in Northern Blot.

Absorbance reading

  • Nanodrop (make sure to set for RNA). Record concentration and A260/A280 ratio. An ideal purity has an A260/A280 between 1.8 and 2.2.

Storage

  • Store at -20°C until needed.
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