cDNA synthesis using superscript II
ATTENTION: This is expensive. One reaction as described here is ca. 6 Euro! Check the quality of your RNA by gel to be sure it is not degraded.
Prepare the annealing mix:
| RNA | 50 ng/µl | 1 µg |
| random hexamers (100 ng/µl) | 25 ng/µl | 5 µl |
| water | add to 20 µl |
Anneal the primers in a thermocycler:
| 1. | 70 °C | 10 min |
| 2. | 25 °C | 10 min |
| 3. | 4 °C | store |
Prepare the enzyme mix:
| 5x First Strand Buffer | 8 µl |
| DDT | 4 µl |
| dNTP (10 mM each) | 2 µl |
| SUPERase inhibitor | 1 µl |
| SuperScript II | 1 µl |
| water | 4 µl |
Prepare this mix as a master mix and add then 20 µl to each reaction. Synthesize the cDNA in a thermocycler:
| 1. | 25 °C | 10 min |
| 2. | 37 °C | 45 min |
| 3. | 42 °C | 45 min |
| 4. | 70 °C | 15 min |
| 5. | 4 °C | store |
Dilute the reaction mix for use in qPCR Add 160 µl water to the 40 µl reaction mix. Use 2 µl per qPCR reaction (that''s equivalent to 10 ng RNA).
Random Primer Hexamers:
Dilute 33.3 µl Random Primer Hexamers (3 µg/µl) with 966.7 µl water to obtain a 100 ng/µl solution.
Materials needed:
SuperScript II (# 18064-014) by Invitrogen Random Primer Hexamers (3 µg / µl (# 48190-011) by Invitrogen SUPERase Inhibitor (# 2694) by Ambion
Commented Protocol: 1. Prepare the annealing mix:
| RNA | 50 ng/µl | 1 µg |
| random hexamers (100 ng/µl) | 25 ng/µl | 5 µl |
| water | add to 20 µl |
Instead of the random hexamers you can use 4 pmole gene-specific primers or 1 µg Oligo(dT)12-18 (each given as total amount for this reaction mix). I prefer the random hexamers, because one synthesis can be used for whatever you want to detect and is not enriching the 3'' ends of the RNA. Furthermore, it just works well.
Add 160 µl water to the 40 µl reaction mix. Use 2 µl per qPCR reaction (that''s equivalent to 10 ng RNA). For some applications it is necessary to get rid of the RNA. Use a RNase incubation followed by a clean up using spin columns.
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