Hi Dr Muhandas

I can suggest you to check two things

1) The dNTP mix containing the DIG labelled dUTP might have gone bad so you need to check it.....you can use the control template (if given) in the kit...

2) If the dNTP mix used by you contains more proportion of DIG labelled dUTP....it can give problem during the amplification as the label is incorporated very frequently..so you can try to dilute the DIG-dNTP mix (as also suggested in the Kit manual)....

bye

Ajay

Try different ratios of DIG-dUTP to dTTP. Lower the amount of DIG label, better the amplification. Instead of 1:3, try 1:5, 1:7 or even 1:10 ratio of DIG to dTTP. When you incorporate labelled NTPs, the intensity of amplification is reduced considerably.

I agree with Mr. Bayram and Mr. Ambaby regarding the use of diluted template. But when i had used the same roche kit for DIG labelling, i got a very sharp and intense band which had a mobility less than the control (unlabelled amplicon).

Thanks all for your expert suggestions and kind help. Today i tried as Mr.Ajay and Mr. Ambaby suggested i diluted my DIG UTP 1:3 times and get two faint bands as Mr.Bayram informed me,and one sharp band from Test DNA, in addition to this i also got sharp band of control without DIG.(lane 1 and 2 in gel are of same gene, 2 is used as unlabelled control). Where as i did not get any band from +ve labelled Control( template+ primer provided by KIT). Now my question is that can i rely on my bands in the absence of amplification of positive control?Gel image is attached( Size of amplicon without labelling should be as Test 1= 700bp, Test 2= 750bp, Test 3= 250 bp)

Thanks BH Kong for your suggestion.Its good to contact them for the problem. Thanks

Hi Dr Muhandas,

did you resolved your problem ?

I am faced exactly to the same difficulty and i have contact Roche who suggest too to dilute the DIG-UTP.

The problem still continu: I have a labeled control tpA which shows no band whereas the unlabeled control tpA shows a strong band.

In addition ma labeled sample shows a faint band. But as you say could we rely on this faint band while the quality control doesn't work?