Daniel:Notebook/PosSequencing/2017-5-31

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Revision as of 18:46, 31 May 2017 by >Djacobse (→‎Protocol)
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Emulsion PCR Test 2 (Started Thursday May 25)

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Yesterday's imaging showed no results. As a quick check I'm going to use SYBR to check the presence of any DNA product. I'll use a positive and negative control to analyze the results more effectively.

Protocol

  1. SYBR Check
    1. Centrifuge sample at 13,000rpm for 1 minute
    2. Remove supernatant except about 10 uL
    3. Add 10 uL SYBR Green; incubate room temp 5 min
    4. Centrifuge sample at 13,000rpm for 1 minute
    5. Remove supernatant except about 10 uL
    6. Add ~40 uL 2X SSC buffer; total volume should be about 50 uL
    7. Add 15 uL to slide for analysis
    ******
    
  2. Empty Beads SYBR Loading (Negative Control)
    1. Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer
    2. Pull down with magnet for 30 seconds and remove supernatant
    3. Add 10 uL nfH2O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes
    4. Pull down with magnet for 30 seconds and remove supernatant
    5. Add 50 uL 2X SSC buffer
    6. Add 15 uL to slide for analysis
    ******
    
  3. dsDNA SYBR Loading (Positive Control)
    1. Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer
    2. Pull down with magnet for 30 seconds and remove supernatant
    3. Add 20 uL Dynabuffer, 15 uL nfH2O, and 5 uL 500 nM barcode 2 oligo
    4. Incubate at RT for 15 minutes
    5. Pull down with magnet for 30 seconds and remove supernatant
    6. Wash with 100 uL dynabuffer
    7. Pull down with magnet for 30 seconds and remove supernatant
    8. Add 10 uL nfH2O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes
    9. Pull down with magnet for 30 seconds and remove supernatant
    10. Add 50 uL 2X SSC buffer
    11. Add 15 uL to slide for analysis