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Daniel:Notebook/HiResChrPaint/2013-7-26
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=FISH= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] Been working on getting FISH slides ready (see [[Daniel:Notebook/HiResChrPaint/2013-7-24|slide prep]]). The key part of this is preparing the hybridization cocktail, everything else is actually pretty simple. The problem I've been having is precipitation. It is easy enough to add the ingredients: Hybridization Cocktail: 2X SSCT, 50% formamide, 10% (w/v) dextran sulfate, 10ug RNAase A, 10-20 pmole probe My recipe has been: {| 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="83" height="30" | Compound | width="85" | Stock Solution Conc. | width="65" | Dilution | width="65" | For 1 mL cocktail |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Formamide | align="center" | 100% | 1:2 | 500 uL |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Dextran Sulfate | 50 % w/v | 1:5 | 200 uL |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | SSCT Buffer | 10X | 1:5 | 200 uL |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | RNAase A | 10 ug/uL | NA | 1 uL |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | nfH20 | NA | NA | 99 uL |} I can add the dextran sulfate, the nfH2O, and the SSCT buffer easily enough, but when I add the formamide something precipitates. However, I scanned through the literature, and I think I have the solution: '''Directions''': Resuspend the pellet in 50% formamide/2Γ SSC/10% dextran sulfate as follows: dissolve the pellet in the appropriate amount of 100% formamide, shake at 37Β°C (can take up to a few hours) and then add an equal volume of 4Γ SSC/20% dextran sulfate. Briefly mix and incubate at 37Β°C for 10 min (see Note 8). '''Note 8:''' Probes containing segments with partial homology to other chromosomes may require higher concentrations of formamide (e.g., 70%) in the hybridization mix in order to reduce unspecific hybridization. As an example, cross hybridization to other chromosomes of centromeric probes that essentially bind to one chromosome can be prevented by hybridization in 70% formamide. -Capitulo, Tecnico Cremer 2008 ''Multicolor 3D-FISH for Interphase Chromosomes'' So, as it turns out, I have been using Dimethylformamide, not regular formamide. The chemical makeups are similar, but their chemical properties are significantly different. For one, dimethylformamide causes swelling of plastic (sound like problems I've had?..). SO, I'll need to readjust here, and make sure to use formamide, not dimethylformamide. Unfortunately, this means the [[Daniel:Notebook/HiResChrPaint/2013-7-24|slides]] prepared Wednesday are more or less useless. Other than that, however, this mistake doesn't have huge implications. Still, sigh...
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