Daniel:Notebook/PosSequencing/2017-7-5: Difference between revisions
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<gallery perrow=2 heights=250px widths=250px mode=packed-hover caption="DRAQ5 DNA staining for 1 pM starting template"> | <gallery perrow=2 heights=250px widths=250px mode=packed-hover caption="DRAQ5 DNA staining for 1 pM starting template"> | ||
File:|Field 1 | File:20170705-PosSequencing-emPCR7.lif_1pM-Field1-SNAP_ch00.jpg|Field 1 | ||
File:20170705-PosSequencing-emPCR7.lif_1pM-Field2-SNAP_ch00.jpg|Field 2 | File:20170705-PosSequencing-emPCR7.lif_1pM-Field2-SNAP_ch00.jpg|Field 2 | ||
File:|Field 3 | File:20170705-PosSequencing-emPCR7.lif_1pM-Field3-SNAP_ch00.jpg|Field 3 | ||
</gallery> | </gallery> | ||
[[Category:20170628]] [[Category:PosSeq]] | [[Category:20170628]] [[Category:PosSeq]] |
Revision as of 23:16, 5 July 2017
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-Field1-SNAP ch00.jpg
Field 1
- 20170705-PosSequencing-emPCR7.lif 1pM-Field2-SNAP ch00.jpg
Field 2
- 20170705-PosSequencing-emPCR7.lif 1pM-Field3-SNAP ch00.jpg
Field 3