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
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