cDNA synthesis in oligo (dT)25 magnetic beads (S1419)
This protocol can be used with 50 - 500 µg of beads (100 µl to 1ml). The following information is for 50 µg of oligo (dT)25 magnetic beads.
- Following the Low-salt buffer wash step, remove and discard low-salt buffer.
- Quickly wash the beads once with cold 1X RT reaction buffer.
- Prepare the following reaction mixture separately and add to the beads immediately following the wash with cold RT buffer.
In a microcentrifuge tube add:
10 µl - 10X RT buffer
16 µl - 10mM dNTP mix
5 µl - 50 U RNase Inhibitor
1µl - 200 U M-MuLV Reverse Transcriptase
68 µl - cold nuclease-free dH2O
100 µl - Total reaction volume
Please note: This reaction can be scaled up (to use a higher number of beads) based on these ratios.
- Incubate at 42°C for at least 1 hour. Gently agitating the beads by hand periodically.
- Wash the beads three times with cold (DNase-free) TE.
- Store immobilized cDNA beads at 1µg/µl in TE at 4°C. Use 10 µl of beads suspension per 50 µl PCR reaction.