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Daniel:Notebook/HiResChrPaint/2014-2-12
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=Multi-Color FISH ([[Daniel:Notebook/HiResChrPaint/2014-2-8|Started 02/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Cot1 Labeling== The size exclusion gel didn't go great last time, so I'm going to try again and have Andrew give me a bit of coaching. ===Size Exculsion Gel=== To separate the fragments, I'll use a size exclusion gel. My protocol is based off [[Arichard:Protocols/MDA amplicon size selection|Andrew's]]. #For each of 4 sample pools ## Place a 0.5 ml tube inside a low binding 1.5 ml tube. Use a 20 G needle to punch a hole in the bottom of the low binding 0.5 ml tube, then UV treat. ## UV treat 400 ul TE buffer for each pool. ## Set up and UV treat a nanosep tube. # Run samples on 5 lane PAGE gels, 250 V for 22 minutes. ##'''Low Mass Ladder''': 10 ul dye, 2 ul low mass ladder. 38 uL 0.5x TBE ##'''Sample''': 40 uL dye, 8 uL Cot1 DNA, 152 uL 0.5x TBE, aliquot 4 lanes of 50 uL each # Stain with 6 uL SYBR Gold; shake for 2 minutes # Size select with scalpel. '''High MW'''- >=400bp; '''Low MW'''- <=200bp # Load gel fragment into 0.5 ml tube (inside 1.5 ml tube) ## Spin for 2 minutes at 12,000 RPM ## Remove and discard 0.5 ml tube. # Add 400 ul TE buffer and incubate shaking for 1 hour at 37 degC. # Load into nanosep tube and spin 3 minutes at 15,000 RPM. # Add 1 mL 100% EtOH, 40uL 3M NaOAc, and 1uL glycoblue ##Incubate overnight at -80C; Continued [[Daniel:Notebook/HiResChrPaint/2014-2-13|tomorrow]] ===Gel Images=== '''Pre-Cut''' [[Image:2014-02-12-Cot1-PreCut.jpg|500px]] '''Post-Cut''' [[Image:2014-02-12-Cot1-PostCut.jpg|500px]] [[Category:HRCP]] [[Category:20140208]] [[Category:Size Exclusion]]
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