Great but loss of activity after one month or so

March 23, 2015

The Ohio State University
Dept of chemistry and biochemistry
Graduate Research associate

Overall

Quality of Results

Ease-of-Optimization

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Company:

Affymetrix Inc.

Product Name:

T4 DNA Ligase

Catalog Number:

70042x

We use USB T4 DNA Ligase to ligate two RNA molecules in a reaction guided by a complimentary DNA splint. Our reaction goes overnight (16hrs) at 37C. Using this method, we are able to get 80% ligation of our RNA which we gel purify.

Experimental Design and Results Summary

Application

Ligation of RNA

Starting Material

5' monophosphorylated piece of RNA, 3' piece of RNA, DNA oligo conplimentary to RNA

Protocol Overview

1. Anneal 2. Incubate RNA and DNA for 3 minutes at 98 C 3. Place at 37 C for 30 min 4. Ligate 5. Incubate annealed RNA/DNA at 37 C 6. Ligase (FC 1U/uL), USB 10x buffer over night. 7. Gel purify products

Tips

Use about 3-4 fold excess of DNA Splint

Results Summary

Expect roughly an 80% yield before gel purification. Clean RNA using Phenol:Cholorform:Isoamyl Alcohol and EtOH Precipitate RNA

Additional Notes

None

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Summary

The Good

Best ligase on the market for this application. Has multiple applications such as ligating DNA into plasmids.

The Bad

Expensive. You must use a lot to get the desired result.

The Bottom Line

This is a great product for use in splinted ligation. It is expensive and must be used as efficiently as possible.

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