Daniel:Notebook/PosSequencing/2017-5-31
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Emulsion PCR Test 2 (Started Thursday May 25)
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
- SYBR Check
- Centrifuge sample at 13,000rpm for 1 minute
- Remove supernatant except about 10 uL
- Add 10 uL SYBR Green; incubate room temp 5 min
- Centrifuge sample at 13,000rpm for 1 minute
- Remove supernatant except about 10 uL
- Add ~40 uL 2X SSC buffer; total volume should be about 50 uL
- Add 15 uL to slide for analysis
- Empty Beads SYBR Loading (Negative Control)
- Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer
- Pull down with magnet for 30 seconds and remove supernatant
- Add 10 uL nfH2O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes
- Pull down with magnet for 30 seconds and remove supernatant
- Add 50 uL 2X SSC buffer
- Add 15 uL to slide for analysis
- dsDNA SYBR Loading (Positive Control)
- Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer
- Pull down with magnet for 30 seconds and remove supernatant
- Add 20 uL Dynabuffer, 15 uL nfH2O, and 5 uL 500 nM barcode 2 oligo
- Incubate at RT for 15 minutes
- Pull down with magnet for 30 seconds and remove supernatant
- Wash with 100 uL dynabuffer
- Pull down with magnet for 30 seconds and remove supernatant
- Add 10 uL nfH2O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes
- Pull down with magnet for 30 seconds and remove supernatant
- Add 50 uL 2X SSC buffer
- Add 15 uL to slide for analysis
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