NSF Soybean Functional Genomics Project
Vodkin Laboratory, University of Illinois |
Reverse Trasncriptase Labeling
and Hybricization with Cy3/Cy5, page 1, 2, 3
Steve Clough/Reena Philip
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| Date of labelling: ________________________ |
Name: _______________________ |
| Slide ID number:________________________ |
Print Date: ____________________ |
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Cy3 label info
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Cy5 label info
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| Tissue |
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| Total or mRNA |
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| Date RNA prepared |
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| Conc of RNA ug/ul |
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| # of ul used |
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| Amt of RNA (ug) |
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(want 2 ug mRNA or 100 ug total RNA per labeling reaction)
| Cy3 |
Cy5 |
Check as added (Cy5 is very senstiive to fluorescent
light so minimize the exposure) |
| ____ |
____ |
1.Mix together: |
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| ____ |
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RNA (or RNA + water) |
8 ul |
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| ____ |
____ |
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oligo dT (18-21 mer, 2.5 ug/ul)
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2 ul |
(Date made: ________) |
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Total volume |
10 ul |
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| ____ |
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2.Heat 70c, 10 minutes |
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| ____ |
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3.Place on ice for 5-10 minutes, then flash spin
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4. Prepare the labeling reaction cocktail:
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Reagent |
vol |
[final] |
Lot/stock # & Date |
| ____ |
____ |
5X Buffer (Gibco) |
6 ul |
1 X |
____________________ |
| ____ |
____ |
DTT (0.1M, Gibco) |
3 ul |
10 mM |
____________________ |
| ____ |
____ |
Low T' dNTP's (Sigma)
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6 ul |
500 uM dACG |
____________________ |
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(2.5 mM dACG, 1.0 mM dT) |
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200 uM dT |
____________________ |
| ____ |
____ |
Cy3- or Cy5-dUTP (1mM) |
3 ul |
100 uM |
____________________ |
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(FluoroLink Cy3-dUTP #53022 or Cy5-dUTP #55022,
AmershamPharmacia) |
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| ____ |
____ |
SuperScript II (200 U/ul) |
2 ul |
13 U/ul |
____________________ |
| ____ |
____ |
Gibco #18064-014 |
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____________________ |
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Total volume |
20 ul per reaction |
| ____ |
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5. |
Add a 20 ul aliquot of the reaction cocktail to 10 ul of RNA/oligo
dT (total 30 ul). |
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| ____ |
____ |
6. |
Finger flip and quick spin. |
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| ____ |
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7. |
Incubate 42c, 1 hour. |
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| ____ |
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8. |
Add 1 ul SuperScript and incubate another hour (2 hour total)
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Lot/Date__________________. |
| ____ |
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9. |
Add 1 ul RNAse A, DNAse-free |
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Lots/Date:__________________. |
| ____ |
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10. |
Add 1 ul RNAse H, DNAse-free (optional) |
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Lots/Date:__________________. |
| ____ |
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11. |
Incubate at 37c for 30 minutes to degrade RNA |
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Post labeling clean up.
| ___ |
If dual labeling, mix together desired probes
at this time prior to Qiagen clean up. Qiagen PCR Purification
kit Cat No. 28104 Lot#:__________________________ |
| ___ |
1. |
Place labeled spin column into collection tube. |
| ___ |
2. |
Add 5 volumes PB Buffer to labeled probe. |
| ___ |
3. |
Transfer to column. Spin 11,000rpm for Eppendorf 5417C for
1 min. |
| ___ |
4. |
Discard flow through. |
| ___ |
5. |
Add 750ul PE Buffer and spin >10,000g for 1 minute. |
| ___ |
6. |
Discard flow through. (optional: repeat steps 5 & 6) |
| ___ |
7. |
Place column back in collection tube and spin to remove traces
of PE Buffer. |
| ___ |
8. |
Transfer column to clean eppendorf tube. |
| ___ |
9. |
Add 50ul EB Buffer. Set one minute. |
| ___ |
10. |
Spin one minute to collect probe. |
| ___ |
11. |
SpeedVac to under desired volume for size of array (takes
about 10-15 min in the speed vac in Room 388 or 25 min in the
speed vac in room 356) |
| ___ |
Optional: Store at -20c until ready to use. |
Hybridization
note: Have slide, treated glass cover slip, hybridization
chamber and the 'hydration papers' ready before preparing the following.
Treat the glass cover slip (22 x40, Corning # 583391) by dipping
briefly in 0.2% SDS, followed by dipping in sterile, filtered water
and let dry at room temperature or blow dry carefully. Touch only
the sides of the cover slip. (The plastic reagent troughs that are
used for the micropipettes are convenient for washing the cover
slips.)
| ___ |
1.Mix: |
Array size: |
22x22 |
22x40 |
Lot/stock # & Date |
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Probe |
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8 ml |
16 ml |
--- |
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20X SSC (sterile filtered) |
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2.45 ml |
4.9 ml |
______________ |
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3% SDS (sterile filtered) |
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1.35 ml |
2.7 ml |
______________ |
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Nucleotide blockers |
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2.2 ml |
4.4 ml |
______________ |
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Total volumes |
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14 ml |
28 ml |
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| ___ |
2. Finger flip and give quick spin |
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| ___ |
3. Denature in boiling water 2 min. |
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| ___ |
Prepare 'hydration papers': Need 2 papers (1 cm x 2 cm filter
paper. Whatman 3MM) per slide
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Need 2 papers (1 cm x 2 cm filter paper. Whatman 3MM) per
slide |
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Place on strip of Parafilm |
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Squirt with water to saturate papers |
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| ___ |
4. Spin 30 seconds at highest speed |
| ___ |
5. Load immediately on array. Should only take a few seconds!
invert slide (array facing down) and place onto cover slip
use pipette tip to gently press cover slip to work bubbles
to edges.
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| by: |
pipette probe onto cover slip (glass cover slip from
Corning #______ ) |
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invert slide (array facing down) and place onto cover
slip |
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use pipette tip to gently press cover slip to work
bubbles to edges. |
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| ___ |
6. Add saturated (not over saturated) 'hydration
papers' about 1 cm from cover slip. |
| ___ |
7. Assemble hybridization chamber quickly. |
| ___ |
8. Incubate for 8-24 hr at 65oC. |
| Nucleotide Blocker: |
Lot/stock # & Date
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| 20 ul |
PolyA DNA (18mer, 10 ug/ul) |
_____________________ |
| 14 ul |
Salmon sperm DNA (2 mg/ul) |
_____________________ |
| 10 ul |
tRNA (5ug/ul) |
_____________________ |
Washing of
slides
Prepare all wash solutions.
You'll need:
4 slide dishes (2 of Wash1, 1 of Wash 2 and 3).
1 slide rack
| Wash 1: |
1X SSC / 0.2% SDS |
Date:__________________ |
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50ml 20XSSC |
Date:__________________ |
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66ml 3% SDS |
Date:__________________ |
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885 ml water |
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| Wash 2: |
0.2X SSC / 0.2% SDS |
Date:__________________ |
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10ml 20X SSC |
Date:__________________ |
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66ml 3% SDS |
Date:__________________ |
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925ml water |
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| Wash 3: |
0.1X SSC |
Date:__________________ |
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5ml 20X SSC |
Date:__________________ |
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995ml water |
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| ___ |
1. |
Disassemble hybridization cassette and remove slide and quickly
do step #2. |
| ___ |
2. |
Hold slide up-side-down in dish of wash solution #1 until
cover slip gently slides off (avoid cover slip scratching the
array). Then transfer to slide rack that is in a dish of Wash
solution1. Repeat using same solution for all slides that need
washing. Cover with aluminum foil to minimize exposure to light. |
| ___ |
3. |
Finish collecting all slides, start shaking Wash 1 dish on
orbital shaker for 15 min. |
| ___ |
4. |
Wash 2 for 15 minutes |
| ___ |
5. |
Wash 3 for 15 minutes |
| ___ |
6. |
Spin dry at 500rpm for 2 minutes, 25c |
Ready to scan!
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