cDNA Synthesis Protocol
(use : TaKaRa cDNA PCR Library Kit Code #6119 and cDNA Synthesis Kit Cat. #6130)
1st strand cDNA synthesis
1. Prepare Master Mix for 1st strand cDNA (always on ice) :
5 x 1st strand synthesis buffer (#6130) = 2 ul
dNTP mixture (#6130) = 0.5 ul
RNA-se inhibitor (#6130) = 0.5 ul
Oligo dT-RA primer (#6119) = 1 ul
M-MLV Reverse Transcriptase* (#6130) = 0.5 ul
____________________________________________
Total = 4.5 ul
2. Prepare template RNA :
1 ug RNA samples + DEPC water (#6130) total volume = 5.5 ul
↓
heat at 65 oC , 5 min
↓
on ice
↓
add Master Mix (4.5 ul) to samples
↓
mix well
↓
put the samples on Thermal Condition
3. Thermal Condition :
Room temperature, 10 min
↓
42 oC, 1 h
↓
80 oC, 5 min
2nd strand cDNA synthesis
4. Prepare Master Mix for 2nd strand cDNA :
Reactan 1st strand cDNA = 10 ul
5 x 2nd strand synthesis buffer (#6130) = 15 ul
dNTP mixture (#6130) = 1.5 ul
DEPC water (#6130) = 44.5 ul
____________________________________________
Total = 71 ul
↓
5. Add E.coli DNA polymerase I (#6130) = 1 ul
6. Add E.coli RNAse H / DNA ligase mixture (#6130) = 1 ul
↓
Thermal Condition :
16 oC, 2 h
70 oC, 10 min
↓
Add T4 DNA polymerase I (#6130) = 2 ul
↓
incubate on 37 oC, 10 min
↓
Add Stop Solution (#6119) = 6 ul
↓
purification of 2nd strand cDNA
Purification of 2nd strand cDNA
Samples 2nd strand cDNA (81 ul)
+ PCI (25:24:1) equal volume (81 ul)
↓
mix well by vortex
↓
15000 rpm, 5 min, 4 oC
↓
take supernatant into new tube
+ CI (24:1) equal volume
↓
mix well by vortex
↓
15000 rpm, 5 min, 4 oC
↓
take supernatant into new tube
+ ammonium acetate 4M (#6119) equal volume
+ isopropanol equal volume
↓
keep on -20 oC, 1 h
↓
mix gently (no vortex)
↓
15000 rpm, 5 min, 4 oC
↓
take pellet
+ 1 ml 70% ethanol (washing)
↓
15000 rpm, 5 min, 4 oC
↓
dry up
↓
+ 5 ul DEPC water (#6130)
↓
ligation of Cassette Adaptor
Ligation of Cassette Adaptor
purification of 2nd strand cDNA samples
+ 2 ul CA (Cassette Adaptor) (#6119)
+ 6 ul ligation solution II
↓
mix well
↓
+ 12 ul ligation solution I
↓
mix well
↓
incubate on 16 oC, 30 min
↓
+ 25 ul ammonium acetate 4M (#6119) (equal volume)
+ 50 ul isopropanol (equal volume)
↓
keep on -20 oC, 30 min
↓
15000 rpm, 5 min, 4 oC
↓
take pellet
+ 1 ml 70% ethanol (washing)
↓
15000 rpm, 5 min, 4 oC
↓
dry up
↓
+ 30 ul DEPC water (#6130)
↓
keep on -20 oC
