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=Multi-Color FISH= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] The goal of my new experiment will be to try FISH using two colors. The samples will be: *Sample: V6S3 stained with 488 and Cot1 stained with 546 *Positive Control: Cot1 stained differentially with 488 and 546 (see below) *Negative Control: DRAQ5 only; may use unlabeled Cot1 DNA ==Cot1 Labeling== Since I now have ULYSIS 488 and ULYSIS 546, I'm going to stain the Cot1 with both to create multi-color banding. My first job will be to size separate the fragments (they are between 50 and 300bp). I'll stain the larger fragments with one dye and the smaller fragments with another. ===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, 40 uL 3M NaOAc, and 1 uL glycoblue ## Incubate overnight at -80C; Continued [[Daniel:Notebook/HiResChrPaint/2014-2-9|tomorrow]] {{GelLanes|Lane1=Low Mass Ladder|Lane2=Cot1|Lane3=Cot1|Lane4=Cot1|Lane5=Cot1}} '''Gel Image''' [[Image:2014-02-08-Cot1-PreCut.jpg|500px]] [[Category:HRCP]] [[Category:20140208]] [[Category:Size Exclusion]]
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