Direct vs Indirect Leabeling - Signal intensity with AminoAllyl Labeling? (Aug/21/2005 )
I'm currently working on increasing brightness of spots for detection and I would like to know if anyone out there has any experience with directl incorporation of fluorescent labeled UTP's vs indirect labeling (such as modified aminoallyl labeling)? Is one better vs the other? Which is better for acheiving brighter spots?
Generally speaking, direct labeling is easier and faster.
Indirect labeling takes longer time because more purification step is needed.
Regarding to the signal intensity and accuracy, Indirect labeling is better than direct labeling.
There are less bias effect in between the dyes.
It has incorporated the AA/AH nucleotide before the NHS fluorescent dyes labeling.
If your goal is to get bright spot, use indirect labeling.
Also make sure use high purity aminoallyl dUTP. Some of the vendors offer aa-dUTP only with 80% purity. Try avoid low quality material. Contact me off line if you need a good source of aa-dUTP.
Use good reagents for coupling reactions. Anything reacts with amine will compete the dyes with your target DNA.
Where can I get aa-dUTP with a high purity? Thanks!