CY-DYE PROBE Synthesis
|Anchored dT25||Amersham, 8 ÂµM, RPK0145, quote 7G-3709, $110|
|or Anchored dT25||or dT23VN, 8 ÂµM, GibcoBRL|
|Random 9-mers||5' NNN NNN NNN 3', 1 Âµg/Âµl, GibcoBRL|
|GFP poly A+ RNA||prepared from plasmid pSP64-GFP|
|SuperScript II||GibcoBRL, 200 U/Âµl, 10000 units, 18064-014, $177|
|5X First Strand Buffer||GibcoBRL, included with enzyme|
|DTT||GibcoBRL, included with enzyme|
|Nucleotides*||Pharmacia, 100mM each, 27-2035-02, $00|
|or Nucleotides*||Promega, 10mmol each, U1330, $75|
|Cy3-dCTP||Amersham, 25nmol, PA53023, quote 1A-3709, $151|
|Cy5-dCTP||Amersham, 25nmol, PA55023, quote 1A-3709, $151|
|RNase Inhibitor||Boehringer Mannheim, 40U/ml, 799 017, $00|
|or Rnasin||Promega, 2500 units, N2511, $82|
|NaOH||Sigma, 2.5M, $00|
|MOPS||Sigma, 2M, $00|
|Glass fiber filter plate||Millipore, MAFB NOB 10, 10/pack, $134|
|Catch Plate||VWR, 622409-108, 50/pack, $00|
|Binding buffer||5.3M Gua-HCl in 150mM Kac pH 4.8 with glacial acetic acid (3-4ml/500ml)|
|Tris-HCl||Sigma, 10mM pH 8.0, T-3038, $00|
|Ethanol||Stores, 80%, $00|
|ProbeQuant G50||Amersham, 27-5335-01, 50/pack, $145|
|Coverslips||VWR, 24x60mm, 48393-106, $15.92|
|Deionized Formamide||Sigma, 100mL, F-9037, $23.60|
|20X SSC||Ambion, 1L, 9763, $30|
|50X Denhardtï¿½s||Fisher, 500g, BP515-5, $42.40|
|10% SDS||Ambion, 500ml, 9822, $40|
|Human CotI DNA||GibcoBRL, 500 units, 15279-011, $75|
|Poly A72||5ï¿½ A72 3ï¿½, GibcoBRL|
|Wash buffers||1XSSC/0.2%SDS, 0.1XSSC/0.2%SDS, 0.1XSSC|
* Nucleotide mix: always use 10mM dGTP/dATP/dTTP and 1mM dCTP; unlabeled dCTP used in 1:1 ratio with Cy-labeled dCTP.
CY-DYE PROBE SYNTHESIS (17 February 2001)
- Mix together:
2 Âµg mRNA
2 Âµl oligo dT23VN (8 ÂµM)
2 Âµl random 9-mers (1 Âµg/Âµl)
1 Âµl GFP mRNA (1 ng/Âµl)
10.5 Âµl total volume
Heat to 70Â°C for 10 minutes.
Chill on ice for 30 seconds.
Add the following:
4 Âµl 5X First Strand Buffer
2 Âµl DTT (0.1 M)
1 Âµl Nucleotide Mix (10 mM G/A/T, 1 mM C)
1 Âµl Cy3- or Cy5-dCTP (1 mM)
0.5 Âµl RNase Inhibitor
Note: when doing multiple reactions, make up premix containing all but Cy-dyes and aliquot 7.5 Âµl premix to each tube before adding Cy-dyes.
Mix contents of tube gently and incubate at RT for 10 minutes.
1 Âµl SuperScriptII
20 Âµl total reaction volume
Mix contents of tube gently and incubate at 42Â°C for 2-3 hours.
Denature cDNA/mRNA hybrid as follows:
Add 2 Âµl 2.5 M NaOH
Incubate at 37Â°C for 10 minutes
Add 10 Âµl 2 M MOPS
Add 200 Âµl binding buffer to probe and mix.
Dispense into glass fiber filter plate.
Place filter plate on top of vacu-system and apply vacuum.
Wash 6x with 200 ml 80% Ethanol.
Place filter plate on top of catch plate along with centrifuge alignment frame.
Do one dry spin to remove residual ethanol (3500 rpm, 5 min).
Add 50 Âµl 10 mM Tris-HCl, pH 8. Incubate 1 minute at RT.
Place filter plate on top of a clean catch plate along with a centrifuge alignment frame and spin (3000 rpm, 5 min).
Repeat with another 50 Âµl Tris-HCl.
Scan probe at OD 260 nm, 550 nm and 650 nm and calculate probe yield, incorporated Cy-dye and specific activity.
Purify probe over G50 column (prespin column (1000x g 1 min), transfer to new tube, apply probe, spin (1000x g 2 min), collect flow-through).
Dry purified probe down in speedvac (1 hour at 50Â°C).
Rinse slides and coverslips by two quick dips in ddH2O, air dry.
Resuspend dried-down probe in 25 Âµl volume of hybridization solution (50% formamide, 5x SSC, 5x Denhardtï¿½s, 0.1% SDS, 100 Âµg/ml poly A72, 100 Âµg/ml human CotI DNA; pass through 0.2 Âµm filter).
Boil probe for 3 minutes, ice 30 seconds, spin briefly.
Combine with appropriate reaction; total volume is now 50 Âµl.
Apply probe to slide and cover with coverslip.
Incubate overnight (14-16 hrs) at 42Â°C in humid chamber.
Preheat wash buffers to 55Â°C.
Remove coverslip by immersing slide in 1x SSC/0.2% SDS.
Wash in 1x SSC/0.2% SDS for 10 minutes at RT.
Wash twice in 0.1x SSC/0.2% SDS at RT for 10 minutes.
Wash twice in 0.1x SSC at RT for 1 minute.
Rinse by two quick dips in ddH2O
Dry with compressed air.
Scan (PMT 600, green (532nm), filter 1; PMT650, red (633nm), filter 2; width 10mm, length 60mm).
PS. Name Fluorescence Solution
Cy3 = green = pink
Cy5 = red = blue