Daniel:Notebook/HiResChrPaint/2013-7-30: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
No edit summary
>Djacobse
No edit summary
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
=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=
=FISH (S3 and S2)=


==Hybridization Cocktail==
==Hybridization Cocktail==


  1. Added 4 uL [[Daniel:Notebook/HiResChrPaint/2013-7-17#Labeling Results|S3]] and [[Daniel:Notebook/HiResChrPaint/2013-7-25#Labeling Results|S2]] for both V4 and V6.
  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
  2. Dried in vaccuum centrifuge
  3. Resuspended in 12.5 uL 100% formamide
  3. Resuspended in 12.5 uL 100% formamide
Line 92: Line 93:
  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. Allow slides to cool to room temperature
  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

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