Daniel:Notebook/HiResChrPaint/2013-7-29: Difference between revisions

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*Followed [[Daniel:Protocols/FISH#Slide Preparation|Original protocol]]
*Followed [[Daniel:Protocols/FISH#Slide Preparation|Original protocol]]


1. Rinse slides in 100% ethanol; allow slides to dry completely
1. Rinse slides in 100% ethanol; allow slides to dry completely
2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media
2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media
3. Add 100 uL of cell suspension per slide roughly in the center
3. Add 100 uL of cell suspension per slide roughly in the center
4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line
4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line
5. Rinse slides briefly in 1X PBS
5. Rinse slides briefly in 1X PBS
6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
7. Rinse briefly in 1X PBS in a coplin jar
7. Rinse briefly in 1X PBS in a coplin jar
8. Incubate for 5 minutes in 2X SSCT
8. Incubate for 5 minutes in 2X SSCT
9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide
9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide
10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C
10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C
11. Slides are typically best used within 1-2 weeks of creation
11. Slides are typically best used within 1-2 weeks of creation
 
=S1 Probe Preparation=
 
Making a new batch of S1 probe, since I have the other three.
 
==PCR Amplification==
 
1. Reaction Table
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
|style="background-color:#FFFF00" align="center" width="65" height="45" | &nbsp;
| width="65" | 20 nM OligoPool
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AP1V41U
| width="65" | 100 uM AP2V4
| width="65" | 100 uM AP1V61U
| width="65" | 100 uM AP2V6
| width="66" | 2 mM aa-dUTP
| width="65" | H20
| width="65" | Total Volume
 
|- style="background-color:#8DB4E2;font-size:12pt" align="center"
| height="15" | per rxn
| align="center" | 0.2
| align="center" | 50
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 2.5
| align="center" | 45.7
| align="center" | 100
 
|- style="font-size:12pt" align="center"
| height="30" | V4S2 (12.5x)
| align="center" | 2.5
| align="center" | 625
| align="center" | 5
| align="center" | 5
| align="center" | 0
| align="center" | 0
| align="center" | 31
| align="center" | 581.5
| align="center" | 1250
 
|- style="font-size:12pt" align="center"
| height="30" | V6S2 (12.5x)
| align="center" | 2.5
| align="center" | 625
| align="center" | 0
| align="center" | 0
| align="center" | 5
| align="center" | 5
| align="center" | 31
| align="center" | 581.5
| align="center" | 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
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===
 
===Nanodrop Results===

Latest revision as of 18:03, 30 July 2013

FISH[edit]

Back to Calendar

FISH Slide Preparation[edit]

1. Rinse slides in 100% ethanol; allow slides to dry completely
2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media
3. Add 100 uL of cell suspension per slide roughly in the center
4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line
5. Rinse slides briefly in 1X PBS
6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
7. Rinse briefly in 1X PBS in a coplin jar
8. Incubate for 5 minutes in 2X SSCT
9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide
10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C
11. Slides are typically best used within 1-2 weeks of creation