Daniel:Notebook/HiResChrPaint/2013-8-5
S1 Probe Preparation (Started 8/2/2013)[edit]
Researched this morning on new ways to improve the FISH protocol. First of all, I noticed that in the supplementary material of Beliveau et al the images are not so crystal clear as the paper (go figure). From the supplements, I see that nucleii seem to have more background than I originally suspected (see thumbnail).
For a good image, the signal should be distinctly above background, but there will likely be background present in the nucleus. Second, also in the thumbnail, it looks like 42 C incubation will be better than 37C. Note that both images are from 16 hour incubation and human stained cells, just as I use. I also found more points, and I will summarize them all here:
- Incubate at 42C instead of 37C; use water bath for this
- Denature using the slide apparatus of the thermocycler we have in lab; can get to 92C easily and more even denaturing than water bath
- Use human cot-1 DNA (at Dr. Zhang's request, would have done this for the last ones but we didn't have any)
- Use fewer probes (10-20 pmole total). The previous FISH experiment used 20 pmol of each probe. Will hopefully reduce background to use less probe
- Use a negative control; maybe hybridize with just the human cot-1 DNA (plus everything else in the hybridization cocktail except probe)
Lambda Exonuclease Digestion[edit]
1. Reaction Table
Sample | V4S1A | V4S1B | V4S1C | V4S1D | V6S1A | V6S1B | V6S1C | V6S1D |
ng/uL | 88.2 | 87.1 | 77.4 | 94.7 | 129.9 | 142.6 | 139.6 | 132.3 |
ug in 25 uL | 2.2 | 2.2 | 1.9 | 2.4 | 3.2 | 3.6 | 3.5 | 3.3 |
uL added | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
uL 10x buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5U/uL) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
uL nfH2O | 17 | 17 | 17 | 17 | 17 | 17 | 17 | 17 |
total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C 3. Pool samples 4. ssDNA column purification, elute with 20 uL 5. Measure Concentration in Nanodrop 6. Dry samples in vaccuum centrifuge
So I made a mistake in the column purification and forgot to SAVE THE FLOW THROUGH despite bold capitalized warnings. I caught myself before messing up V6, but I lost V4.
Nanodrop Results[edit]
ng/uL | ug in 15 uL | |
V4S1 | 362.2 | 5.4 |
So I have around 5 ug of DNA left in V6S1. By the math I have a little more than 5 ug left, but I think I can still get away with just 1 tube of dye.
Dye Coupling[edit]
Following ARES Protocol
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Resuspend DNA in 5 uL nfH20 and add 3 uL sodium bicarbonate buffer 3. Denature samples for 5 minutes at 95C, then snap cool using ice box 4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 5. Add 2 uL dye to sample 6. Incubate in the dark for 1 hour 7. Add 15 uL nfH20 to sample 8. Centri-Sep purification
Labeling Results[edit]
ng/uL DNA | pmol/uL dye | ug DNA in 20 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V6S1 | 144.7 | 6.1 | 2.9 | 5.4 | 1.1 | 1.4 | 72 |
Great run. Just over 1 fluorophore per probe.