|
Preparation of Fluorescent DNA Probe from HUMAN mRNA or Total RNA using Direct Incorporation (Max Diehn/Ash Alizadeh protocol; 3/15/01)
I. Preparing fluoresenctly labeled cDNA (probe):
- To anneal primer, mix 2ug of mRNA or 50-100mg total RNA with 4ug of a regular or anchored oligo-dT primer in a total volume of 15.4 ul:
Cy3
Cy5
mRNA (1
x
y l
(2 mg of each if mRNA, 50-100mg if total RNA)
Oligo-dT (4
1
1
(Anchored: 5'-TTT TTT TTT TTT TTT TTT TTV N-3')
ddH2O (DEPC)
to 15.4
to 15.4
Total volume:
15.4
15.4
oC for 10 min and cool on ice.
Reaction mixture
l
. . . . .
Unlabeled dNTPs
Vol.
Final conc.
5X first-strand buffer*
6.0
dATP (100 mM)
25 uL
25 mM
0.1M DTT
3.0
dCTP (100 mM)
25 uL
25 mM
Unlabeled dNTPs
0.6
dGTP (100 mM)
25 uL
25 mM
Cy3 or Cy5 (1 mM, Amersham)
3.0
dTTP (100 mM)
10 uL
10 mM
Superscript II (200 U/uL, Gibco BRL)
2.0
ddH2O
15 uL
Total volume:
14.6
Total volume:
100 uL
* 5X first-strand buffer: 250 mM Tris-HCL (pH 8.3), 375mM KCl, 15mM MgCl2)
oC for 1 hr. l SSII (RT booster) to each sample. Incubate for an additional 0.5-1 hrs. ml of 0.1N NaOH, 2mM EDTA and incubate at 65-70oC for 10 min. If starting with total RNA, degrade for 30 min instead of 10 min. ml of 0.1N HCl. l l l lll l g/l)l g/l)
- Heat to 65
- Add 14.6 mL of reaction mixture each to Cy3 and Cy5 reactions:
- Incubate at 42
- Add 1
- Degrade RNA and stop reaction by addition 15
- Neutralize by addition of 15
- Add 380ml of TE (10mM Tris, 1mM EDTA) to a Microcon YM-30 column (Millipore). Next add the 60ml of Cy5 probe and the 60ml of Cy3 probe to the same microcon. (Note: If re-purification of cy dye flow-through is desired, do not combine probes until Wash 2.)
- WASH 1: Spin column for 7-8 min. at 14,000 x g. mg of Cot1 human DNA (20mg/ml, Gibco-BRL), 20mg polyA RNA (10mg/ml, Sigma, #P9403) and 20mg tRNA (10mg/ml, Gibco-BRL, #15401-011). Spin 7-10 min. at 14,000 x g. Look for concentration of the probe in the microcon. The probe usually has a purple color at this point. Concentrate to a volume of less than or equal to the volume listed in the "Probe & TE" column in the table below. These low volumes are attained after the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane. Make sure not to dry the membrane completely!
Select the appropriate row from the table:
Cover Slip Size (mm) Total Hyb Volume (ml) Probe & TE (ml) 20x SSC (ml) 10% SDS (ml) 22 x 22 15 12 2.55 0.45 22 x 40 25 20 4.25 0.75 22 x 60 35 28 5.95 1.05
*20x SSC: 3.0 M NaCl, 300 mM NaCitrate (pH 7.0)
- Ready washes in 250 ml chambers to 200 ml volume as indicated in the table below. Avoid adding excess SDS. The Wash 1A chamber and the Wash 2 chambers should each have a slide rack ready. All washes are done at room temperature.
-
Wash Description Vol (ml) SSC SDS (10%)
- Blot dry chamber exterior with towels and aspirate any remaining liquid from the water bath.
- Unscrew chamber; aspirate the holes to remove last traces of water bath liquid.
- Place arrays, singly, in rack, inside Wash I chamber (maximum 4 arrays at a time). Allow cover slip to fall, or carefully use forceps to aid cover slip removal if it remains stuck to the array. DO NOT AGITATE until cover slip is safely removed. Then agitate for 2 min.
- Remove array by forceps, rinse in a Wash II chamber without a rack, and transfer to the Wash II chamber with the rack. This step minimizes transfer of SDS from Wash I to Wash II.
- Wash arrays by submersion and agitation for 2 min in Wash II chamber, then for 2 min in Wash III (transfer the entire slide rack this time).
- Spin dry by centrifugation in a slide rack in a Beckman GS-6 tabletop centrifuge at 600 RPM for 2 min
- Scan arrays immediately.
- WASH 2: Remove flow-through and add 450 ul TE and spin for 7-8 min. at 14,000 x g. It is a good idea to save the flow trough for each set of reactions in a separate microcentrifuge tube in case Microcon membrane ruptures.
上一篇：Amino-allyl Reverse Transcription 下一篇：Protocol for Reverse Transcription and Amino-allyl Coupling of RNA
共2页: 上一页 1 [2] 下一页 |
