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=S1 Probe Preparation ([[Daniel:Notebook/HiResChrPaint/2013-8-2|Started 8/2/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] 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). [[Image:Fish-hybridization-temp-compare.png|600px|thumb]] 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 [[Daniel:Notebook/HiResChrPaint/2013-8-1|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== 1. Reaction Table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="80" height="15" | Sample | width="65" | V4S1A | width="65" | V4S1B | width="65" | V4S1C | width="65" | V4S1D | width="65" | V6S1A | width="65" | V6S1B | width="65" | V6S1C | width="65" | V6S1D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 88.2 | align="center" | 87.1 | align="center" | 77.4 | align="center" | 94.7 | align="center" | 129.9 | align="center" | 142.6 | align="center" | 139.6 | align="center" | 132.3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 25 uL | align="center" | 2.2 | align="center" | 2.2 | align="center" | 1.9 | align="center" | 2.4 | align="center" | 3.2 | align="center" | 3.6 | align="center" | 3.5 | align="center" | 3.3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL added | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL 10x buffer | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="45" | uL Lambda Exonuclease (5U/uL) | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nfH2O | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 |} 2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C 3. Pool samples 4. [[Daniel:Protocols/ssDNA_Column|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=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="65" height="15" | |style="font-weight:bold" width="65" | ng/uL |style="font-weight:bold" width="65" | ug in 15 uL |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V4S1 | align="center" | 362.2 | align="center" | 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== Following [[Daniel:Protocols/AlexaDyeCouple#ARES|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 β₯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. [[Daniel:Protocols/CentriSep|Centri-Sep purification]] ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="65" height="30" | |style="font-weight:bold" width="65" | ng/uL DNA |style="font-weight:bold" width="65" | pmol/uL dye |style="font-weight:bold" width="65" | ug DNA in 20 uL |style="font-weight:bold" width="71" | pmol/uL DNA |style="font-weight:bold" width="71" | dye/probe |style="font-weight:bold" width="71" | dye/100 bp |style="font-weight:bold" width="71" | Base:Dye Ratio (:1) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V6S1 | align="center" | 144.7 | align="center" | 6.1 | align="center" | 2.9 | align="center" | 5.4 | align="center" | 1.1 | align="center" | 1.4 | align="center" | 72 |} Great run. Just over 1 fluorophore per probe.
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