Atto488 NT Labeling Kit for Generating Probes for DNA FISH

September 08, 2015

BlueskyReddit
Yale University
Cell Biology
Associate Research Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

Jena Bioscience

Product Name:

Atto488 NT Labeling Kit

Catalog Number:

PP-305S-488

We have generated a cell line based system where we have introduced a single copy transgene to study gene transcription. To confirm that our transgene is single copy we wanted to carry out DNA FISH. As the size of the transgene is small we expected the DNA FISH signal to be weak. We chose this product to generate a directly labeled probe instead of other immuno-based DNA FISH kits to increase signal to noise ratio by avoiding background generated by non-specific binding of antibodies.

Experimental Design and Results Summary

Application

Generating probes of DNA FISH

Starting Material

2 KB DNA

Protocol Overview

1. Fix cells with 4% PFA 2. Permeabilize with 0.5% triton in 1PBS 3. Wash with PBST (0.05% tween 20) 4. Prehybridize with 50% formamide in 2X ssc 5. Hybridize with probe for overnight in presence of formamide and non-specific binding DNA 6. Wash and acquire images

Tips

Always check DNA probe size by running on agarose gel.

Results Summary

We saw weak signals for our transgene DNA FISH, which we believe is mostly is due to small DNA target. Probes generated from a BAC vector had much stronger signal.

Additional Notes

None

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Summary

The Good

Convenient, direct labeling decreases background signal

The Bad

Might need to standardize the probe synthesis when targeting smaller targets for DNA FISH

The Bottom Line

I would recommend this product for probe generation for DNA and RNA FISH. It had much better signal:noise ratio as compared to other immuno-based probe generation kits. Moreover the protocol was shorter.

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