Daniel:Notebook/PosSequencing/2017-4-28
Emulsion PCR (Started Yesterday)
Images and Analysis
Field 4
- 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
- 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