Skip to content

Common Lab Protocols

Jump to bottom Edit New page
kubu4 edited this page Oct 20, 2016 · 37 revisions

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)
  • TE
  • 100% EtOH
  • 70% EtOH
  • phenol:chloroform:IAA (25:24:1)
  • 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)

#####Personal Protective Equipment (PPE):

  • Gloves

#####Equipment:

####Procedure #####Total Time: 3 days #####Cost/sample:

Add a custom footer
Home
Informatic Protocols
Lab Notebook Best Practices
Lab Protocols
Clone this wiki locally