Saturday 05/18/13

11:00 A – 7:30 P, 8:30 P – 12:30 A

Probe making

• cast 3 15% TBE Urea PAGE gels 0.75mm 15 well
• cast 1 15% TBE Urea PAGE gel 1.5mm 5 well

PAGE gel

• lanes: ULR ladder 1:2 in TBE, Y 1:5 pre-digest, K pre-digest, AA pre-digest, AB pre-digest, Y 1:20 post-digest and EtOH prec, K post-digst “, AA post “, AB post “, ULR ladder 1:2

PCR

Emulsion PCR:

• Blue Adapter-primer + Blue Nicksite-primer + orig LC library
• Yellow Adapter-primer + Yellow Nicksite-primer + orig LC library
• Ubx Adapter-primer + Ubx Nicksite-primer + Ubx emPCR product

Regular PCR

1. Blue Adapter-primer + Blue Nicksite-primer + orig LC library
2. Yellow Adapter-primer + Yellow Nicksite-primer + orig LC library
3. Ubx Adapter-primer + Ubx Nicksite-primer + Ubx emPCR product
4. Black Adapter-primer + Black outside-Nick-site-primer + K emPCR product
5. Black RP + Black outside Nick-site-primer + K emPCR product
6. Black RP + Black Nick-site-primer + K emPCR product
7. Blue S1-adapter + blue Nicksite-primer + blue emPCR product
8. Yellow S1-adapter + yellow Nickstie-primer + yellow emPCR product
9. Black S2-adapter + black Nicksite-primer + black emPCR product
10. Green S1-adapter + green Nicksite-primer + green emPCR product

* Run each in 2 separate tubes (control for early misamplifiaction/tube variance)

PCR (15x)
150 µl 10x buffer
30 µl 10 mM dNTP mix
15 µl KAPA Taq
1275 µl ddH2O

1 µl 100 µM “R” primer
1 µl 100 µM “F” primer
1 µl 1 ng/µl complex DNA library (or 1 µl 0.01 µM library)

Gel

STORM of Ph

• staining all 6 wells of sample plate 1 with WGA-488, old 1:10 dilution increased to 1:500, incubated 15 mins on coverglass mounts and washed 2x 15 min+ in PBT (none of these samples have 488 channel used). Aim is to avoid background coming in from nuclei/DAPI when using 405 excitation laser.
• set up to image wt ph rb-647.
• highly variable cell to cell staining
• images kinda look more like Ph-wt.