cDNA synthesis protocol.

rabc010 at S1.CXWMS.AC.UK rabc010 at S1.CXWMS.AC.UK
Fri Oct 24 11:04:16 EST 1997


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.



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