T7 RNA polymerase amplification protocol (Elva 7/30/03)
1.
Combine 1-5 µg total RNA (for small samples use everything you got) with 2
µl of T7 (5) primer (100 ng/µl) in vol of 12 µl (sequence 5”AAATTAATACGACTCACTATAGGGAGACCACA(T)21-3’)
2.
Incubate 70C for 10 min, crash cool on ice, quick spin down
3.
Make mix, add 7µl per tube:
4 µl 5X BRL super
1 µl 0.1M DTT
1 µl RNasin (Promega)
1 µl 10 mM dNTP
4.
Add 1 µl Superscript III (invitrogen), incubate 50C 2 hr to O/N
5.
Place on ice
6.
Make 2nd strand reaction mix, add 125.5 µl to each tube
92.5 µl H20
30 µl 5X 2nd strand buffer (ED)
3 µl 10 mM dNTP
6.5
Make enzyme mix, add 4.5 µl to each tube on ice
0.5 µl RNase H (Amersham, 5 unit/µl)
3 µl DNA pol 1 (10 unit/µl, NEB)
1 µl E. coli DNA ligase (10 unit/µl, NEB)
7.
Incubate 16C 3 hr or O/N
8.
Phenol/chloroform extract using PLG tubes
Pellet PLG tubes, add 2nd strand reaction (150 µl)
Add equal vol of phenol/chloroform (DNA/RNA), shake by hand (I
usually add slightly less vol ~135 µl because the PLG tubes get very full
when doing the chloroform extraction and can spill)
Spin 5 min RT, 14K rcf
Add equal vol chloroform (~135 µl), repeat as above
Remove upper phase to new tube
9.
Add 2 µl LPA (10 µg), 0.5 vol 7.5 MNH4Oac (75 µl), mix
10.
Precipitate with ethanol (450 µl), incubate 30 min at –20C (not O/N, free
nucleotide will start to precipitate upon O/N incubation)
11.
Spin 25 min to pellet, max speed
12.
Wash with 0.5 ml 75% ethanol, spin, remove all drops
13.
Resuspend in 8 µl 10 mM Tris pH7.4 or dH20, 10 min RT or brief incubation
at 37C to ensure that the cDNA is resuspended
14.
Proceed to Ambion’s T7 Megascript kit, set up reaction at RT (never put these
reactions on ice)
15.
Make mix (at RT!), add 10 µl to each tube:
8 µl rNTP (2 µl of each A/G/U/C
2 µl 10X reaction mix (heat at 37C to make sure in solution)
16.
Add 2 µl enzyme mix to each tube, incubate 37C for 5 hr
17.
Add 1 µl DNase I (included in kit), incubate 15 min at 37C
18.
Stop with 15 µl stop solution (included in kit)
19.
Bring up to 100 µl with 65 µl dH20 for phenol/chloroform extraction
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Prepare PLG tubes by quick spin, add 100 µl phenol/chloroform (RNA)
Add reaction mixture, mix by hand
Spin RT 5 min at 14K rcf
Add 100 µl chloroform, proceed as above
Add 2 µl LPA, mix and precipitate with 100 µl isopropanol
Incubate 30 min at –20C (I don’t precipitate O/N here because you get a lot of
junk that precipitates, you can store the pellet in the 75% ethanol wash if
necessary)
Spin 25 min, max speed cold, (you should see a big pellet)
Wash with 75% ethanol, 0.5 ml (can store here at –20C, see step 25)
Remove all fluid droplets, resuspend in 20 µl dH20 or 10 mM Tris pH 7.4
Heat 5-10 min at 37-50C, spec 0.7 µl and/or run on gel
For long term storage, freeze on dry ice and store –80C
** Notes, you can use Superscript II in place of SSIII but then use twice the amount
of 0.1 M DTT and do the reaction for 1 hr at 42C (follow Invitrogen’s instructions).
If you have a lot of free nucleotide after the cDNA precipitation, you can clean up the
cDNA by using a small round Millipore filter.
I usually resuspend in water unless I am going to store the sample for a long time.