There were some people who had problem with cDNA synthesis using

Smart cDNA synthesis kit (Clontech). Here is the protocol, I am

using for cDNA synthesis. I will be glad if it is useful for anyone.

Good luck.

Narendra Kaushik
Neuroscience & Psycological Medicine,
Imperial College at Charing Cross Hospital,
Fulham Palace Road, London W6 8RF UK
email. nkaushik at hgmp.mrc.ac.uk

Protocol for cDNA synthesis.

Synthesis of Ist strand cDNA on the beads.

Use magnetic beads to isolate mRNA, dont elute mRNA from
the beads leave it on. This will make life bit more easier as you
will see.

Reverse Transcription of mRNA.

Final volume 20 ul
dH2O X ul
5X buffer 4.0 ul
DTT ( 0.1 M) 2.0 ul
dNTP (10mM) 1.0 ul
mRNA 0.5 ug (on the beads).
Total 19.0 ul
SupersriptII 1.5 ul (Life technologies).

Incubate 42oC/1hr. Stir the beads occasionally.

Put the tube into the magnetic strand and discard the buffer, 2x wash
with TE (10mM Tris pH 8.0, 1mM EDTA).

A-Tailing.

Total vol 30 ul.

dH2O x ul
5x Terminal transferase buffer 6 ul
2mM dATP 3 ul
Terminal transferase 10 U

Incubate 20-30 min/37oC. Incubation time and conc. of dATP are

not important. Length of A tail will not affect PCR as anchored
oligo dTVN primer is used.
2x Wash the beads with TE and 1x wash with PCR buffer.

PCR.
use 50ng of mRNA equivalent beads in 100ul reaction for PCR.

PCR primer. I use OligodT primer with CUA that is helpful in

cloning using Uacil DNA glycosylase cloning system (pAMP10,
Life technologies) without any restriction enzyme digestion, you can
incorporate restriction enzyme site into your primer .

CUA CUA CUA CUA TTT TTT TTT TTT TTT VN

CDNA 50ng (on the beads)

10xPCR 10 ul (Klentaq buffer,with 3.5 mM
MgCl2).
dNTP 2mM 10 ul
Primer (10 uM) 10 ul (1 uM final)
Klentaq1 0.5 ul
dH2O > 100 ul

94oC/5min x1
94oC/30s
60oC/2min
72oC/4min
25 cycles
72oC/10min
Dont use too many cycles to amplify.
Size select by purifying fron agarose gel(1.5%, TAE) > 500bp and
clone into the vector
Comparison of Reaction Assemblies and Protocols

Bio-Rad Laboratories' iScript cDNA Competitor I's First-Strand cDNA

Synthesis Kit Synthesis System
To a 0.2 ml tube, add: To a 0.2 ml tube, add:

5X cDNA synthesis kit buffer Oligo(dT) primers 

iScript enzyme mixture Random hexamers 
Nuclease-free water Buffer 
RNA sample MgCl2 
dNTPs 
DTT 
RNase inhibitor 
Reverse transcriptase 
Nuclease-free water 
RNA sample

Next, program the thermal cycler to run the following protocol:

5 min at 25°C  5 min at 65°C 

30 min at 42°C  Remove, quick chill on ice 
5 min at 85°C  Add additional items 
Hold at 4°C Mix and incubate 2 min at 42°C 
Add reverse transcriptase 
Return reaction to thermal cycler 
50 min at 42°C 
15 min at 70°C 
Hold at 4°C 
RNase H digestion, 20 min at 37°C

Total time, including setup and reaction:

45 min >110 min

Diethylpyrocarbonate (DEPC), also called diethyl dicarbonate (IUPAC name), diethyl
oxydiformate, ethoxyformic anhydride, or pyrocarbonic acid diethyl ester, is used in
the laboratory to inactivate theRNase enzymes from water and other laboratory utensils.
It inactivates the RNases by the covalent modifications of the histidine residues. DEPC
cannot be used with Tris buffer or HEPES since they inactivate DEPC by reacting with it.
In contrast it can be used with PBS or MOPS. A handy rule is that enzymes or chemicals
which have active -O:, -N: or -S: cannot be treated with DEPC to become RNase-free as
DEPC reacts with these species. Furthermore DEPC degradation products can inhibit in
vitro transcription.

Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 1 hour at 37°C
and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC
in this manner yields CO2, H2O and EtOH. Higher concentrations of DEPC are competent
of deactivating larger amounts of RNAse but remaining traces or byproducts may inhibit
further biochemical reactions such as in vitro translation. Further on, chemical
modification of RNA such as carboxymethylation is possible when traces of DEPC or its
byproducts are present, resulting to reduced usage of RNA even after buffer exchange
(after precipitation).

DEPC treated water for use in a laboratory

DEPC treated (and therefore RNase-free) water is used in handling of RNA in the
laboratory, to reduce the risk of RNA being degraded by RNases.

DEPC derivatization of histidines is also used to study the importance of histidyl residues
in enzymes. Modification of histidine by DEPC results in carbethoxylate derivates at the
N-omega-2 nitrogen of theimidazole ring. DEPC modification of histidines can be
reversed by treatment with 0.5M hydroxylamineat neutral pH