Daniel:Notebook/PosSequencing/2017-4-28
Emulsion PCR (Started Yesterday)[edit]
Images and Analysis[edit]
Field 4[edit]
- 20170428-emPCR2 w2GFP4.jpg
Alexa 488; barcode oligo v1
- 20170428-emPCR2 w4Cy3-4.jpg
Cy3 image (Note: No Cy3 was used, therefore there shouldn't be a Cy3 signal)
- 20170428-emPCR2 w3Cy5-4.jpg
Cy5 image; barcode oligo v3
- 2017-04-28-emPCR-Field4-Overlay-Cy3-GFP-Cy5.jpg
Dye overlays; Cy3-white; A488-green; Cy5-Red
- 20170428-Field4Overlay-Measured.png
Dye overlays, individual bubbles measured as 2.5 um each (2 A488, 1 Cy5)
Field 4 had the best results and is worth a summary. There are 5 fluorescent spots. Not counting Cy3, which shouldn't be there and has extra signal that is not present in the other channels. Two of the molecules are too large to be a single bead, which should be ~1 um. However, 3 of the signals are that size. Two have A488 signal and one has Cy5 signal.
Other Fields[edit]
- 2017-04-28-emPCR-Field1Overlay.jpg
Field 1 overlay; Note that this one is brightfield/A488 only. The Cy5 channel was weak on this one, and Cy3 wasn't measured
- 2017-04-28-emPCR-Field2-Overlay-Cy3-GFP-Cy5.jpg
Field 2 image; Cy3-white; A488-green; Cy5-Red; two small patches near the center may be the right size, and only contain the A488 signal
- 2017-04-28-emPCR-Field3-Overlay-Cy3-GFP-Cy5.jpg
Field 3 image; Cy3-white; A488-green; Cy5-red; red/green overlap is yellow; Both signals are too large here. The large one at the bottom also contains signal in every channel
- 2017-04-28-emPCR-Field5-Overlay-Cy3-GFP-Cy5.jpg
Field 6 image; strong Cy3 signal everywhere. Also, most green dots also have a strong yellow presence, meaning both Cy5 and A488 were present. However, almost all the dots are too large to be a single bead anyway; inconclusive
Conclusion[edit]
In conclusion I don't think the experiment had much conclusive results. There do appear to be several beads that monoclonal and are the right size. There is also significant Cy3 signal that shouldn't be there and Cy5 seems weak. Only 1 Cy5 bead was suggested in field 4, whereas 2 A488 beads were in a 4 and possibly another 2 in field 2. Recommend repeating without the emulsions and with only single fluorophores (1 each) and a mixed channel to show what can happen when the beads are mixed/unmixed. Basically, a bunch of positive controls for comparison.