Daniel:Notebook/PosSequencing/2017-7-5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
Line 45: Line 45:


<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)

Back to Calendar

Protocol

  1. Template-bead binding; do this twice, once with no template
    1. Suspend 1 uL (10 ug, ~10M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
    4. 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
    5. Wash twice with 100 uL dynabuffer
    6. Resuspend beads in 50 uL TE buffer
  2. DRAQ5 staining
    1. Take 5 uL aliquots and add 43 uL nfH2O and 2 uL DRAQ5 (1:10000)
  3. Slide preparation
    1. Add 15 uL of aliquot to a slide
    2. Add a #1.5 coverslip and seal with nail polish; wait approximately 20 minutes before imaging

Results