P-CAGE-25010 - P-CAGE-25010

Type
labeling
Description
Concentrate RNA using microcon.

Take 100-150 µg RNA in 80 µl H2O, put in microcon column. Centrifuge 4min at 14000g. Turn column in clean tube. Centrifuge for 3 min at 1000 g. Measure concentration (0.5 µl in 300 µl 10 mM Tris pH8)


Reverse transcription.

Prepare two 200 µl PCR tubes according to the recipe below:

100 µg of total RNA + 2 µl oligo dT21 mer (1 mg/ml) + enough water to achieve 13.4 µl final volume. Heat 5 min at 70oC in PCR machine. Leave at RT for 5 min. Prepare mix according to the following recipe:

6 µl 5x SuperScript II Buffer +

3 µl 0.1 M DTT +

0.6 µl dNTPS (25 mM dATP, dGTP, dTTP; 10 mM dCTP) +

2 µl SuperScript II Reverse Transcriptase (Invitrogen)+

2 µl RNase Inhibitor (Invitrogen).

Add 13.6 µl mix per tube.

Add: 3 µl/tube Cy3-dCTP (1 mM) for each sample. Mix well and incubate at 42oC for 2 hr in PCR machine.


End of probe preparation.

Pool the two tubes together (corresponding Cy3 and Cy5 samples).

Add: 2.65 µl 25 mM EDTA + 3.30 µl 1 M NaOH.

Incubate 10 min at 65oC in PCR machine.

Add: 3.3 µl 1 M HCl + 5 µl 1 M Tris-HCl, pH 6.8.

Purify probe with Qiagen MinElute PCR purification kit (#28004)

Mix 5 volumes (370 µl) buffer PB with 1 volume of probe (74 µl).

Add 3 µl 3M NaAc. pH 5.2 and mix. Transfer to column.

Spin 1 min at ≥ 10,000 x g. Discard flowthrough.

Add 750 µl buffer PE to column. Spin 1 min at ≥ 10,000 x g. Discard flowthrough. Spin 1 additional min. Transfer column to new 1.5 mL tube. Add 10 ml of buffer EB to the center of the membrane. Wait 1 min then centrifuge for 1 min. Repeat the elution step : ~20 ml eluate. Quantify using Nanodrop (2 µl/20).

Mix in a a 200 µl PCR tube:

labeled probe 18 µl +

H2O 53.2 µl +

SSC 20X 15 µl +

SDS 10% 3.8 µl +

tRNA 2 µg/µl 10 µl +

Total 100 µl.

Parameters
Amount of nucleic acid labeled, Amplification, Used Label
Links
All experiments using protocol P-CAGE-25010: (E-ATMX-36, E-ATMX-37, E-ATMX-9)