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RNA Isolation

Standard Operating Protocol (SOP)

Written 20150616 by Sam White.

Reagents:
  • RNAzol RT (MRC: RN 190) (SOP)
  • Isopropanol (2-propanol) (SOP)
  • 75% Ethanol made with 0.1% DEPC-treated H2O
  • 0.1% DEPC-treated H2O (SOP)
  • ice
Personal Protective Equipment (PPE):
  • Gloves
  • Safety glasses
  • Long sleeves
  • Fume hood
Equipment:
  • Pipettes (10 - 1000uL)
  • Filtered pipette tips
  • Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S)
  • Microfuge (capable of 12,000g)
  • Disposable mortar and "pestle" tubes (Fisher: K7495200090)

Procedure

Total Time: ~ 1.5 - 2.5hrs
Cost/sample: ~ $1.50
  1. Read the SOPs for each of the reagents used in this protocol.
  2. Read the manufacturer's RNAzol RT protocol for Total RNA Isolation.
  3. Read this protocol.
  4. Verify sufficient quantities/availability of reagents/equipment.
  5. Wear clean gloves and change gloves frequently.
  6. Aliquot 500uL of RNAzol RT to pestle tubes and store on ice.
  7. Transfer tissue to pestle tubes containing RNAzol RT.
  8. Homogenize immediately with disposable pestle.
  9. Immediately add additional 500uL of RNAzol RT to pestle tube.
  10. Vortex 15s.
  11. Add 400uL of 0.1% DEPC-treated H2O.
  12. Vortex 15s.
  13. Incubate at room temperature (RT) for 15mins.
  14. Centrifuge 12,000g for 15mins @ RT.
  15. Transfer 750uL of supernatant (do not disturb pellet) to sterile 1.7mL snap-cap tube. (Discard remaining liquid in RNAzol RT Hazardous Waste container in fume hood. Leave old tube open in fume hood over night and then discard in regular trash.)
  16. Add 1 volume of isopropanol.
  17. Vortex 5s.
  18. Incubate @ RT for 15mins.
  19. Centrifuge 12,000g for 10mins @ RT.
  20. Discard supernatant; do not disturb pellet.
  21. Add 400uL of 75% ethanol, centrifuge 4,000g for 3mins @ RT.
  22. Repeat steps 20 and 21 one time.
  23. Repeat step 20.
  24. Centrifuge 4,000g for 1min @ RT.
  25. Removal residual ethanol.
  26. Immediately resuspend pellet in appropriate volume of 0.1% DEPC-H2O (volume is dependent upon pellet size, but 50uL is usually sufficient).
  27. Keep sample on ice for short-term storage (i.e. no more than 2hrs) or store @ -80C.

DNase RNA

Standard Operating Protocol (SOP)

Written 20150616 by Sam White.

Reagents:
Personal Protective Equipment (PPE):
  • Gloves
Equipment:
  • Pipettes (10 - 200uL)
  • Filtered pipette tips
  • 0.5mL snap-cap microfuge tubes (Genesee: 22-178A)
  • Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S)
  • Microfuge (capable of 10,000g)
  • Thermal cycler, water bath, or heating block capable of 37C.
  • ice

Procedure

Total Time: ~ 1.5 - 2.0hrs
Cost/sample: ~ $2.00
  1. Read the manufacturer's Turbo DNA-free rigorous protocol.
  2. Read this protocol.
  3. Verify sufficient quantities/availability of reagents/equipment.
  4. Wear clean gloves and change gloves frequently.
  5. Thaw RNA on ice.
  6. Reaction volume will be 50uL.
  7. Transfer desired quantity of RNA to a 0.5mL tube, but no more than 2ug and no more than 44uL.
  8. Adjust RNA volume in 0.5mL tube to 44uL with nuclease-free H2O (supplied in kit).
  9. Add 0.1 volumes (5uL) of 10x Turbo DNase Buffer.
  10. Add 1uL of Turbo DNase. Mix by gently flicking tube. If needed, spot spin for no more than 2s to collect liquid from sides of tube.
  11. Incubate @ 37C for 1hr.
  12. Repeat steps 10 & 11.
  13. Vortex DNase Inactivation reagent for 10s.
  14. Add 0.2 volumes (10.2uL) of DNase Inactivation to the RNA.
  15. Incubate @ RT for 2mins with occasional mixing.
  16. Place 0.5mL tube in an empty 1.7mL tube and centrifuge 10,000g for 1.5mins @ RT.
  17. Transfer supernatant to sterile 1.7mL tube. Do not transfer any inactivation reagent. If some is transferred, repeat steps 16 & 17.
  18. Keep sample on ice for short-term storage (i.e. no more than 2hrs) or store @ -80C.

Reverse Transcription

Standard Operating Protocol (SOP)

Written 20150702 by Sam White.

Reagents:
Personal Protective Equipment (PPE):
  • Gloves
Equipment:
  • Pipettes (10 - 1000uL)
  • Filtered pipette tips
  • 0.5mL snap-cap microfuge tubes (Genesee: 22-178A)
  • Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S)
  • Thermal cycler, water bath, or heating block capable of 37C OR 42C.
  • vortexer
  • ice

Procedure

Total Time: ~ 1.5 - 2.0hrs
Cost/sample: ~ $1.50

IMPORTANT: A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used in this protocol are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

  1. Read the manufacturer's protocol.

  2. Read this protocol.

  3. Verify sufficient quantities of reagents and samples before beginning.

  4. Wear clean gloves.

  5. Thaw all RNA and reagents on ice. Prepare all reactions on ice.

  6. Transfer 1ug of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.

  7. Add 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.

  8. Heat samples at 70C for 5 min in thermal cycler, heating block, or water bath.

  9. Place samples on ice IMMEDIATELY.

  10. Make Master Mix:

    Per Reaction

    • 5 uL 5x Buffer (M-MLV RT Buffer)

    • 1.25 uL 10mM dNTPs

    • 0.5 uL M-MLV RT per ug of RNA

  11. Mix well by flicking; do not vortex.

  12. Add 6.75uL of master mix to each reaction.

  13. Mix by pipetting; do not vortex.

  14. Incubate @ 42C for 1hr for oligo dT primers OR @ 37C for random primers.

  15. Heat inactivate @ 95C for 3 min.

  16. Spot spin and store @-20C.

qPCR

Standard Operating Protocol (SOP)

Written 20150702 by Sam White.

Reagents:
  • SsoFast EvaGreen Supermix (BioRad: 172-5203)
  • Primer working stocks (10uM)
  • DNase-free H2O (NanoPure H2O)
Personal Protective Equipment (PPE):
  • Gloves
Equipment:

Procedure

Total Time: ~ 2.0 - 4.0hrs
Cost/sample: ~ $0.42

  1. Read the manufacturer's protocol.

  2. Read this protocol.

  3. Verify sufficient quantities of reagents and samples before beginning.

  4. Wear clean gloves.

  5. Prepare master mix. Be sure to make a master mix volume that will accommodate the following: all of your samples, two water (i.e. no template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. A single reaction is shown below:

    Component Volume(uL) Final Concentration
    2x SsoFast EvaGreen Supermix 10 1x
    Forward Primer (10uM) 0.5uL 0.2uM
    Reverse Primer (10uM) 0.5uL 0.2uM
    Water Variable Use to bring reaction volume up to 20uL (including template)

  6. Distribute appropriate amount of master mix (volume of master mix + template = 20uL) to white PCR plate.

  7. Add template.

  8. Cap with optical PCR caps.

  9. Spin plate for 1min @ 3000g.

  10. Put plate in real-time PCR machine.

  11. Recommended cycling parameters (40 cycles) are listed below. They may need to be changed to accommodate your specific primers/samples. See manufacturer's protocol for recommendations.

    Step 1 - 98C 2mins

    Step 2 - 98C 5secs

    Step 3 - 60C 5secs

    Step 4 - Plate read

    Step 5 - Got to Step 2 39 more times

    Step 6 - Melt curve 65C - 95C, increment 0.5C, wait 2secs, plate read.

MeDIP (Methylated DNA Immunoprecipitation)

Standard Operating Protocol (SOP)

Written 20130513 by Sam White

Adapted from:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763296/

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013100

Reagents:
  • DNAzol (Molecular Research Center) (SOP)
  • TE (SOP)
  • Ice cold 100% ethanol (SOP)
  • 70% ethanol (SOP)
  • phenol:chloroform:IAA (25:24:1) (SOP)
  • 5x MeDIP Buffer (50mM Na2HPO4, 700mM NaCl, 0.25% Triton-X 100)
  • anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b)
  • Protein A/G Plus Agarose beads (Santa Cruz Biotech)
  • 3M sodium acetate (pH = 5.2)
  • MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS)
  • Proteinase K (SOP)
Personal Protective Equipment (PPE):
  • Gloves
Equipment:
  • Refrigerated microfuge
  • Room temp microfuge
  • Sonicator

Procedure

Total Time: 3 days
Cost/sample:

Notes:

  • Both MeDIP Buffers should be made up from liquid stocks of each individual component, as each individual component are common molecular biology stock reagents that all lab members should have at their benches. Do NOT attempt to make the MeDIP Buffers by weighing out and dissolving powdered chemicals for each individual component; it cannot be done accurately with the small quantities required.

  • This is a multi-day procedure. Read through the protocol thoroughly to plan your time properly.

  • Isolate gDNA according to DNAzol protocol (Molecular Research Center), but resuspend final DNA pellet in TE or H2O. (Initial incubation with Proteinase K is dependent on tissue type and available time to perform procedure.)

  • Quantify gDNA yield and quality. Procedure requires a minimum of 6ug, but more can't hurt.

DAY 1

  1. Fragment gDNA with sonicator to a target size of 500bp.
  2. Run 250ng of fragmented on an 2% agarose gel to verify successful fragmentation. Additionally, the fragmentation should be verified/quantified on an Agilent Bioanalyzer, if possible.

If fragmentation is successful, proceed to Step 3.

  1. Bring fragmented gDNA sample to a volume of 350uL with TE.
  2. Heat sample at 95C, 10mins and immediately place on ice for 5mins.
  3. Add 100uL of 5x MeDIP Buffer (50mM Na2HPO4, 700mM NaCl, 0.25% Triton-X 100), 45uL of TE, and 5uL (5ug) of anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b). Incubate O/N, @ 4C, rotating end-over-end.

DAY 2

  1. Wash appropriate volume of Protein A/G Plus Agarose beads (Santa Cruz Biotech; need 20uL per sample) with 1x MeDIP Buffer.
  2. Mix stock Protein A/G Plus Agarose beads well and transfer needed volume to clean tube. Pellet beads by spinning 1000g, 2mins, 4C. Discard supernatant. Resuspend beads in 1mL of 1x MeDIP Digestion Buffer. Repeat one time. Final resuspension in 1x MeDIP Buffer. Final resuspension volume is 40uL per sample. Add 40uL of resuspended Protein A/G Plus Agarose beads to each sample and continue incubation with end-over-end rotation @ 4C for 2hrs.
  3. Pellet the Protein A/G beads 1000g, 2mins, 4C.
  4. Remove and retain supernatant (to retain unmethylated DNA).
  5. Wash beads with 1mL 1x MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS) a total of three times. Save supernatant after each wash in its own tube (this will simplify DNA precipitation later on).
  6. Resuspend beads in 250uL MeDIP Digestion Buffer.
  7. Add 70ug of Proteinase K and incubate 24hrs @ RT with end-over-end rotation. Note: The source protocols say to incubate the Proteinase K digest @ 55C. However, we don't have a means to do so, since we need a rotator to keep the agarose beads in suspension. According to various sources, Proteinase K retains >80% of it's enzymatic activity between 20C - 50C. So, allow the digest to run longer (24hrs) than recommended (O/N).

DAY 3

  1. Add a volume of phenol:chloroform:IAA (25:24:1) equal to your sample volume, vortex thoroughly, and spin 16,000g, 10mins, RT.
  2. Transfer aqueous phase to clean tube. If sample had cloudy interphase repeat Step 1 until interphase is no longer cloudy.
  3. Precipitate your various DNAs.
  4. Add 1/10th volume of 3M sodium acetate (pH = 5.2), 2.5 volumes of ice cold 100% ethanol, mix thoroughly, and incubate @ -20C for at least 20mins. Note: If expecting low yields, addition of 20ug of glycogen can help improve recovery.
  5. Pellet DNA by spinning samples at 16,000g, 20mins, 4C.
  6. Discard supernatant and wash pellet with 1000uL 70% EtOH.
  7. Re-pellet DNA by spinning samples at 16,000g, 20mins, 4C.
  8. Discard supernatant, pulse spin, remove residual supernatant, and briefly air dry pellet for 5mins at RT.
  9. Resuspend methylated DNA in 50uL TE. Resuspend each unmethylated DNA fraction in 25uL and then pool.
  10. Quantify DNAs.

DNA Isolation from Water Filters

Standard Operating Protocol (SOP)

Written 20170227 by Sam White

Based on DNeasy Blood & Tissue Kit protocol

Reagents:
Personal Protective Equipment (PPE):
  • Gloves
Equipment:
  • Heating block/incubator at 56C
  • Room temp microfuge
  • 2mL microfuge tubes (Genesee: 22-283A)

Procedure

Total Time: 2 days
Cost/sample: ~$2.75

NOTES:

  • This protocol uses twice the volume of the Qiagen-supplied reagent, Buffer AL. Depending on needs, additional Buffer AL may be required in order to process an entire kit's worth of samples.

  • This protocol uses twice the volume of the Qiagen-supplied reagent, . Depending on needs, additional Proteinase K may be required in order to process an entire kit's worth of samples.

  • This protocol is written for DNA isolation from Graham-positive bacteria. Graham-negative bacteria requires Buffer ATL.

  • This protocol has not been tested for isolation of DNA from eukaryotic organisms captured on water filters.

DAY 1

  1. Read the SOPs for each of the reagents used in this protocol.

  2. Read the manufacturer's DNeasy Blood & Tissue Kit protocol

  3. Read this protocol.

  4. Add 400uL of Buffer AL to tube(s).

  5. Cut water filter(s) into ~13 equal size strips (assuming filters are ~50mm diameter).

  6. Insert strips into tube(s) with collection-side facing out.

  7. Add 50uL of Proteinase K to tube(s).

  8. Vortex at high speed for ~30 seconds.

  9. Incubate O/N at 56C.

DAY 2

  1. Add 400uL of 100% EtOH to tube(s).

  2. Vortex at high speed for ~30 seconds.

  3. Follow manufacturer's protocol for remainder of procedure.

  4. Elution volume of 100uL of Buffer AE is recommended, but should be adjusted empirically for a given sample type.

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