problem with TNT coupled invitro transcription/ - (Apr/16/2004 )

i am trying to express SARS subgenomic mRNAs invitro.i am using TNT@T7 coupled invitro transcription translation kit .i cloned subgenomic mRNAs containing l(eader sequence) in pBluscript IIKS (+). correct oreintation and reading frames were varified by DNA sequencing. plasmids were extracted with qiagene minispin kit and plasmids were linearised with appropriate enzymes and purifed with phenol:chlo:iso and chlo: iso , precipitated with 0.3 Na-acetate and washed with 70% ethanol,air dried and resuspended in nuclease free water.1-2ug DNA was used in 50ul reaction i got band of expected size in positive control provided with kit but i did not got any band in case of subgenomic mRNAs( i am also using subgenomic mRNA encoding spike protein that is known to be expressed in SARS virus infected cells) . can any one tell me what is wron , where is the problem and how can i overcome this .....

i appriciate it very much


snawar

Snawar

You have stated that your control is working fine. There is something in your test sample DNA that is causing the problem. Test this by adding 2 -8ul of heat denatured test sample to the kit control. If you get no band now, you have an inhibitor originating in your test sample.

Good luck and God Bless