Daniel:Notebook/HiResChrPaint/2013-7-30: Difference between revisions
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2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar | 2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar | ||
3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes | 3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes | ||
4. | 4. Remove slides and allow to cool to room temperature | ||
5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip | 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip | ||
6. Invert slides on to cocktail-covered coverslips; seal with rubber cement | 6. Invert slides on to cocktail-covered coverslips; seal with rubber cement |
Revision as of 16:31, 31 July 2013
S1 Probe Preparation
Making a new batch of S1 probe, since I have the other three.
PCR Amplification
1. Reaction Table
20 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V41U | 100 uM AP2V4 | 100 uM AP1V61U | 100 uM AP2V6 | 2 mM aa-dUTP | H20 | Total Volume | |
per rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 2.5 | 45.7 | 100 |
V4S1 (12.5x) | 2.5 | 625 | 5 | 5 | 0 | 0 | 31 | 581.5 | 1250 |
V6S1 (12.5x) | 2.5 | 625 | 0 | 0 | 5 | 5 | 31 | 581.5 | 1250 |
2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold
qPCR Results
This PCR didn't work. Will have to mull over why and try again tomorrow.
FISH (S3 and S2)
Hybridization Cocktail
1. Added 4 uL S3 and S2 for both V4 and V6. 2. Dried in vaccuum centrifuge 3. Resuspended in 12.5 uL 100% formamide 4. Added 12.5 uL 4X SSCT + 20% Dextran Sulfate
FISH Slide Preparation
Following original FISH protocol:
1. Allow stored slides to warm to room temperature 2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar 3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes 4. Remove slides and allow to cool to room temperature 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip 6. Invert slides on to cocktail-covered coverslips; seal with rubber cement 7. Allow the rubber cement to air-dry for 5 minutes at room temperature 8. Denature for 2.5 minutes at 92C; keep humid 9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C