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
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
6. Measure concentrations in nanodrop


===qPCR Results===
===qPCR Results===


===Nanodrop 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]

Back to Calendar

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]

File:2013-7-30-S1.png

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