cDNA synthesis using superscript II
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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 |
1. | 70 °C | 10 min |
2. | 25 °C | 10 min |
3. | 4 °C | store |
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 |
1. | 25 °C | 10 min |
2. | 37 °C | 45 min |
3. | 42 °C | 45 min |
4. | 70 °C | 15 min |
5. | 4 °C | store |
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:
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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.
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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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