Daniel:Notebook/PosSequencing/2017-7-5: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=emPCR 7-Strand Separation (Started Wednesday June 28)= Back to Calendar ==Protocol== <ol> <li...") |
>Djacobse m (→Protocol) |
||
Line 19: | Line 19: | ||
</ol> | </ol> | ||
<li>Slide preparation</li> | <li>Slide preparation</li> | ||
<ol type="A"> | |||
<li>Add 15 uL of aliquot to a slide</li> | <li>Add 15 uL of aliquot to a slide</li> | ||
<li>Add a #1.5 coverslip and seal with nail polish; wait approximately 20 minutes before imaging</li> | <li>Add a #1.5 coverslip and seal with nail polish; wait approximately 20 minutes before imaging</li> | ||
</ol> | </ol></ol> | ||
[[Category:20170628]] [[Category:PosSeq]] | [[Category:20170628]] [[Category:PosSeq]] |
Revision as of 20:09, 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)