Making cDNA usingSuperScript III Reverse Transcriptase

Collect RNA using “Preparation of Total TC RNA” protocol but instead dissolving RNA pellet in ddw

The following 20 ul reaction volume can be used for 10 pg – 5 ug of total RNA or 10 pg– 500 ng of mRNA.

To a nuclease-free microcentrifuge tube add:
1 ul of 50 uM oligo(dT)50; or 200-500 ng of oligo(dT)12-18
10 pg – 5 ug total RNA; or 10 pg – 500 ng mRNA . 1 ul 10 mM dNTP mix at neutral pH
Sterile, distilled water to 13 ul

Heat mixture to 65 degrees C for 5 min. and incubate on ice for at least 1 min.

Collect the contents of the tube by brief centrifugation and add:
4 ul 5X First-Strandbuffer (Invitrogen)
1 ul 0.1 M DTT
1 ul RNaseOUT
1 ul of Superscript IIIRT (200 units/ul)

Mix by pipetting gently up and down.

Incubate at 50 degrees C for 30-60 min.

Inactivate the reaction by heating to 70 degrees C for 15 min.

Remove RNA complementary to cDNA by adding 1 ul (2 units) of E. coli Rnase H and incubate at 37 degrees C for 15 min.

Older protocol– Amplification of cDNAs

Preparation of cDNA:

10 µl:
1 µl 10xTKE
20 µg total cellular RNA
10 pmole oligo-dT24(or 0.5 pmole of an mRNA-specific oligo) .

95C, 1'
50C, 15'
Ice.

+40 µl:
10 µl 5xM-MLV RT buffer
10 µl 2.5 mM dNTPs
400 units M-MLV RT
37C, 1h.

1 µl RNaseA (10mg/ml)
37C, 1h.

Spin through SephadexG50 column.
+ 50 µl TE Extract withPhenol:CHCl3
+10 µl 3M NaAc (pH6.0)
10 µg glycogen
275 µl 100% EtOH
Ppt., wash, dry
Dissolve in 50 µl Tebuffer (should be enough for 50-200 PCR reactions)

First round PCR:

20 µl:
2 µl 10xPCR buffer (forAmpli-Taq Gold)
1.5 µl 25 mM MgCl2
1 µl 2.5 mM dNTPs
0.5 µl cDNA
5 pmole oligo 1
5 pmole oligo 2
0.25 µl Ampli-Taq Gold

95C, 3 min
35 cycles: 95C,1 min
60C, 1 min
72C, 2 min per kb product
72C, 5 min
5C, for ever

(Annealing temperatureshould be optimized for every reaction).

Gel purify PCR product.

For cDNAs that are long or have high GC:

20 µl:
2 µl "buffer3"
2 µl 2.5 mM dNTPs
0.5 µl cDNA
5 pmole oligo 1
5 pmole oligo 2
0.25 µl Extend-longpolymerase mix (Boehringer)

For cDNAs that are abundant and short:

20 µl:
2 µl 10xPfu (Buffer inStratagene catalog)
2 µl 2.5 mM dNTPs
0.5 µl cDNA
5 pmole oligo 1
5 pmole oligo 2
0.25 µl Pfu DNApolymerase

Second round PCR:

50 µl:
5 µl 10xPfu (Buffer inStratagene catalog)
5 µl 2.5 mM dNTPs
0.5 µl First-round PCRproduct
10 pmole oligo 1
10 pmole oligo 2
0.5 µl Pfu DNApolymerase

95C, 3 min
35 cycles: 95C,1 min
60C, 1 min
72C, 4 min per kb product
72C, 5 min
5C, for ever

(Annealing temperatureshould be optimized for every reaction).

Gel purify PCE product.

Cut and clone as described in "Cloning" protocol

Buffers:

10xTKE:
100 mM Tris-HCl pH7.0
400 mM KCl
1 mM EDTA