Daniel:Notebook/PosSequencing/2017-7-5
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emPCR 7-Strand Separation (Started Wednesday June 28)
Protocol
- Template-bead binding; do this twice, once with no template
- Suspend 1 uL (10 ug, ~10M beads) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
- Add 5 uL 500 nM (25 pmol total, 0.5X excess) dsDNA and 15 uL nfH2O per sample to bead solution; incubate at RT for 15 min; for NT sample add 20 dynabuffer/20 nfH2O
- Wash twice with 100 uL dynabuffer
- Resuspend beads in 50 uL TE buffer
- DRAQ5 staining
- Slide preparation
- Add 15 uL of aliquot to a slide
- Add a #1.5 coverslip and seal with nail polish; wait approximately 20 minutes before imaging
- Take 5 uL aliquots and add 43 uL nfH2O and 2 uL DRAQ5 (1:10000)
Results
- 20170705-PosSequencing-emPCR7.lif BlankBeads-Field1-SNAP ch00.jpg
Field 1
- 20170705-PosSequencing-emPCR7.lif BlankBeads-Field2-SNAP ch00.jpg
Field 2
- 20170705-PosSequencing-emPCR7.lif BlankBeads-Field3-SNAP ch00.jpg
Field 3
- 20170705-PosSequencing-emPCR7.lif dsDNA-Field1-SNAP ch00.jpg
Field 1
- 20170705-PosSequencing-emPCR7.lif dsDNA-Field2-SNAP ch00.jpg
Field 2
- 20170705-PosSequencing-emPCR7.lif dsDNA-Field3-SNAP ch00.jpg
Field 3
- 20170705-PosSequencing-emPCR7.lif 100pM-Field1-SNAP ch00.jpg
Field 1
- 20170705-PosSequencing-emPCR7.lif 100pM-Field2-SNAP ch00.jpg
Field 2
- 20170705-PosSequencing-emPCR7.lif 100pM-Field3-SNAP ch00.jpg
Field 3
- 20170705-PosSequencing-emPCR7.lif 1pM-Field2-SNAP ch00.jpg
Field 2