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    RNA Extraction Protocol PDF

    Uploaded by

    Ade Oktiviyari

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    RNA Extraction Protocol PDF

    Uploaded by

    Ade Oktiviyari
    73 views2 pages

    Original Title

    RNA extraction protocol.pdf

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    © © All Rights Reserved

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    Copyright:

    © All Rights Reserved
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    Download as pdf or txt
    73 views2 pages

    RNA Extraction Protocol PDF

    Uploaded by

    Ade Oktiviyari

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    You are on page 1of 2

    RNA extraction protocol Date:

    Samples:

    Preparations
    - Syringes 2 ml with 19G and 23 G needles
    - 15 ml Isopropanol at -20 °C
    - 15 ml EtOH 75% in DEPC water at -20 °C
    - Precool centrifuge to 4 °C and always double-check settings
    - Clean hood and bench space

    Sample lysis and phase separation


    Perform all steps in the hood

    1. Keep samples on ice


    2. Add 1ml Trizol in the hood
    3. Homogenize sample with 19G, then with 23G needle
    4. Incubate 5 min in hood
    5. Prepare tubes with 200 µl Chloroform
    6. Add Trizol content to the tubes
    7. Shake vigorously
    8. Incubate 3 min
    9. Centrifuge 15 min 12,000g at 4 °C

    RNA Isolation
    1. Prepare new tubes with 500 µl Isopropanol
    2. Take supernatant (ca. 550 µl), don’t take to much (protein layer at interphase) and add to isopropanol
    3. Keep on ice for 2 h or at 4 °C over night
    4. Centrifuge for 30 min 12,000 g at 4 °C
    5. Set the heatblock to and thaw DEPC water
    6. Discard the supernatant
    7. Wash with 1 ml 75% EtOH
    8. Vortex briefly, then centrifuge 5 min 7,500 g at 4 °C
    9. Discard the supernatant
    10. Place upside-down to dry briefly (not much more than for 5 min)

    RNA solubilization
    1. Add 45 µl DEPC water to the pellet
    2. Heat for 15 min at 55 °C
    3. Resuspend sample

    DNase treatment
    Use Ambion DNA-free™ DNA Removal Kit
    Reaction setup:

    1 rxn Master mix


    Sample 44 µl
    Buffer 5 µl
    DNase 1 µl
    total 50 µl

    1. Incubate 30 min at 37 °C
    2. Add 5 µl DNase Inhibitor
    3. 2 min incubation, vortex every 30 s
    4. Spin down 10,000 g 1.5 min
    5. Transfer supernatant to new tube

    RNA quantification
    1. Use 1x DNA-free kit buffer as blank
    2. Measure RNA by loading 2 µl in duplicate
    3. Check for protein contamination (260/280, should be > 1.7) and for other contamination (eg phenols)
    (260/230, also >1.7)

    Sample ng/µl 260/280 260/230

    Reverse transcription
    Using High-Capacity cDNA Reverse Transcription Kit by Applied Biosystems

    Reaction setup:

    1 rxn Master mix


    Sample 20 µl
    Buffer 10x 4 µl
    25x dNTP Mix 1.6 µl
    (100 mM)
    10x RT Random 4 µl
    Primers
    DEPC water 8.4
    Enzyme 2 µl
    total 40 µl

    Use Martins 3-Step protocol on Thermocycler, ca. 2.5 h

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