Daniel:Notebook/HiResChrPaint/2014-2-8
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Multi-Color FISH
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 Andrew's.
- For each sample pool:
- 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.
- Mix and UV treat 1 ml 100% EtOH, 40 ul 3 M NaAc, and 1 ul GlycoBlue in a 1.5 ml tube.
Protocol
Size selection
- Pool similar samples (keep bacteria separate from mammalian samples), up to 2 samples per pool. Match libraries with similar intensities on the analysis gel to avoid excessive imbalance between libraries.
- Run whole pools on 5 lane PAGE gels, 250 V for 22 minutes. Use 10 ul dye, 2 ul low mass ladder. Use additional 0.5x TBE to equalize volumes between all pools.
- Add 6 ul SYBR Gold to tray with ~1 cm 0.5x TBE, mix well.
- Incubate gel 2 minutes in tray with SYBR Gold on shaker.
- Size select between 300 and 600 bp with scalpel. Load gel fragment into 0.5 ml tube (inside 1.5 ml tube).
- Spin gel in tube-in-tube setup 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.