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

~ oleh adrianna pada November 29, 2009.

Tinggalkan Balasan

Isikan data di bawah atau klik salah satu ikon untuk log in:

Logo WordPress.com

You are commenting using your WordPress.com account. Logout / Ubah )

Gambar Twitter

You are commenting using your Twitter account. Logout / Ubah )

Foto Facebook

You are commenting using your Facebook account. Logout / Ubah )

Foto Google+

You are commenting using your Google+ account. Logout / Ubah )

Connecting to %s

 
%d blogger menyukai ini: