Daniel:Notebook/HiResChrPaint/2014-2-14: Difference between revisions
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=Multi-Color FISH ([[Daniel:Notebook/HiResChrPaint/2014-2-8|Started 02/08/2014]])= | |||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[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:2014-02-14-Cot1-PreCut.jpg|500px]] | [[Image:2014-02-14-Cot1-PreCut.jpg|500px]] | ||
'''Post-Cut''' | |||
[[Image:2014-02-14-Cot1-PostCut.jpg|500px]] | [[Image:2014-02-14-Cot1-PostCut.jpg|500px]] |
Revision as of 20:51, 14 February 2014
Multi-Color FISH (Started 02/08/2014)
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 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 tomorrow
Gel Images
Pre-Cut
File:2014-02-14-Cot1-PreCut.jpg
Post-Cut