Daniel:Notebook/HiResChrPaint/2014-2-12: Difference between revisions

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# Add 1 mL 100% EtOH, 40uL 3M NaOAc, and 1uL glycoblue
# Add 1 mL 100% EtOH, 40uL 3M NaOAc, and 1uL glycoblue
##Incubate overnight at -80C; Continued [[Daniel:Notebook/HiResChrPaint/2014-2-13|tomorrow]]
##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]]

Latest revision as of 18:20, 2 July 2014

Multi-Color FISH (Started 02/08/2014)[edit]

Back to Calendar

Cot1 Labeling[edit]

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[edit]

To separate the fragments, I'll use a size exclusion gel. My protocol is based off Andrew's.

  1. For each of 4 sample pools
    1. 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.
    2. UV treat 400 ul TE buffer for each pool.
    3. Set up and UV treat a nanosep tube.
  2. Run samples on 5 lane PAGE gels, 250 V for 22 minutes.
    1. Low Mass Ladder: 10 ul dye, 2 ul low mass ladder. 38 uL 0.5x TBE
    2. Sample: 40 uL dye, 8 uL Cot1 DNA, 152 uL 0.5x TBE, aliquot 4 lanes of 50 uL each
  3. Stain with 6 uL SYBR Gold; shake for 2 minutes
  4. Size select with scalpel. High MW- >=400bp; Low MW- <=200bp
  5. Load gel fragment into 0.5 ml tube (inside 1.5 ml tube)
    1. Spin for 2 minutes at 12,000 RPM
    2. Remove and discard 0.5 ml tube.
  6. Add 400 ul TE buffer and incubate shaking for 1 hour at 37 degC.
  7. Load into nanosep tube and spin 3 minutes at 15,000 RPM.
  8. Add 1 mL 100% EtOH, 40uL 3M NaOAc, and 1uL glycoblue
    1. Incubate overnight at -80C; Continued tomorrow

Gel Images[edit]

Pre-Cut

File:2014-02-12-Cot1-PreCut.jpg

Post-Cut

File:2014-02-12-Cot1-PostCut.jpg