Daniel:Notebook/PosSequencing/2017-7-5
emPCR 7-Strand Separation (Started Wednesday June 28)[edit]
Protocol[edit]
- 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[edit]
- 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
Discussion[edit]
It's not exactly easy to spot unless you have a fancy computer, so here's the gist. The blank beads samples show that without DNA the DRAQ5 doesn't stain the beads, and there isn't enough autofluorescence at that wavelength to look like much. When you do max projection (which all of these are) and adjust the thresholds to similar values as with the samples, the resulting signal is not next to the dots. The dsDNA positive control on the other hand, has the signal overlap with the beads, although not on all samples. This makes sense, however, as those beads are generally under packed (50% of theoretical maximum was added), meaning a simple Poissonian predicts that a lot of them will have little signal. Some, however, will and do have the signal.
The 100 pM samples have good signal, although I will note that on the samples the beads tend to stick together more often. The 1 pM samples on the other hand, show no real signal. So the 1 pM sample did not have enough amplification.