Daniel:Notebook/HiResChrPaint/2013-7-30: Difference between revisions
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=S1 Probe Preparation= | =S1 Probe Preparation= | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | |||
Making a new batch of S1 probe, since I have the other three. | Making a new batch of S1 probe, since I have the other three. | ||
Line 68: | Line 70: | ||
vii. 72C 2 min | vii. 72C 2 min | ||
viii. 16C hold | viii. 16C hold | ||
===qPCR Results=== | ===qPCR Results=== | ||
=== | [[Image:2013-7-30-S1.png|600px]] | ||
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 (~20 pmol) [[Daniel:Notebook/HiResChrPaint/2013-7-17#Labeling Results|S3]] and [[Daniel:Notebook/HiResChrPaint/2013-7-25#Labeling Results|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== | |||
[[Daniel:Protocols/FISH|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 |
Latest revision as of 17:53, 5 August 2013
S1 Probe Preparation[edit]
Making a new batch of S1 probe, since I have the other three.
PCR Amplification[edit]
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[edit]
This PCR didn't work. Will have to mull over why and try again tomorrow.
FISH (S3 and S2)[edit]
Hybridization Cocktail[edit]
1. Added 4 uL (~20 pmol) 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[edit]
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