P-FPMI-52 - P-FPMI-52

Type
labeling
Description
Reverse Transcription Reaction to make cDNA from mRNA


1.Follow instructions for Qiagen Labelstar kit (28902).

2.Adjust the volume of 1 ul total RNA to 18 ul.

3.Add 2 ul Denaturation solution. Mix and centrifuge briefly.

4.Incubate at 65•C for 5 min and ice immediately. Centrifuge briefly.

5.Prepare a fresh master mix, the following is recipe for 1X reactions: (5 ul 10X Buffer RT, 5 ul dNTP mix (5mM dATP, 5mM dGTP, 5mM dCTP, 1.3mM dTTP), 5 ul oligo-dT primer, 0.5 ul Rnase inhibitor, 10 ul Rnase-free water, 2.5 ul LabelStar Reverse Transcriptase)

6.In Control reaction (will be labelled silver during hybridization), add 1x master mix, 20 ul denatured RNA template (control), 2 ul Fluorescein-labeled dUTP (Enzo cat#42831)7.Mix well, spin briefly.

8.Incubate at 37•C for 2 hours.

9.Add 1 ul dTTP, mix and centrifuge briefly.

10.Incubate at 37•C for 1 hour.

11.Add 2 ul Stop solution to each tube.

12.Pool both labeling reactions. Mix well and centrifuge.

13.Add 530 ul Buffer PB, mix by gently vortexing. Apply to MinElute Spin Column. Centrifuge at 12000X for 1min. Discard flow-through.

14.Wash with 750 ul Buffer LS. Centrifuge at 12000X for 1min. Discard flow-through.

15.Wash with 750 ul Buffer PE. Centrifuge at 12000X for 1min. Discard flow-through.

16.Centrifuge at 12000X for 1min. Discard flow-through.

17.Replace collection tube with a fresh 1.5ml collection tube. Add 42 ul Rnase-free water (Sigma-aldrich). Incubate for 1 minute, Centrifuge at 12000X for 1minute.

18.Proceed to hybridization protocol.

Parameters
Amount of nucleic acid labeled, Amplification, Label used
Links
All experiments using protocol P-FPMI-52: (E-FPMI-2, E-FPMI-3)