severe troubles with Titan RT-PCR kit from Boeringer

Harald Sigmund harald at mol.univie.ac.at
Fri Jul 18 18:47:00 EST 1997


Hi,

I experiencing severe troubles using the Titan One-Tube RT-PCR system
from Boehringer. The problem is that there is no product to be seen on an 
agarose gel stained with ethidium bromide. 

Control reactions using a plasmid containing the target sequence 
with different primer pairs, different dNTP batches and different Taq 
batches (Gibco) proofed that the primers as well as dNTPs and Taq are ok, 
and that the raction works fine witrh the plasmid. 

However, the control reaction using the Titan kit resulted in no product 
bands to be seen on the gel, not even using thew plasmid.

The total volume of the reaction was 25 and 50 µl, we used both SDS-free 
RNA as well as RNA precipitated from an SDS-containing buffer and washed 
SDS-free, no reagents were DEPC-treated. The total amount of RNA-template 
was 2.5 µg total RNA, and the DNA template was 1 µl of an 1:3000 diluted 1 
µg/µl plasmid stock. The expected PCR product is in the range of 370 to 
440 bp length.

temperature program was as following:

50°/30 min

94°/2 min

94°/ 30 sec
62°/ 30 sec
68°/ 45 sec
40 cycles

68°/7 min

6°/infinite

I would be glad if ther eis anyone who can provide possible solutions of 
this problems: although we got a new kit (but from the same batch), the 
Boeringer guy assured me that there are no problems reported so far for 
this system. I hope he is right...

Harald


  




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