Title: Plus A
Author: Travis C. Glenn1,2, [Kenneth L. Jones1,2, Myriam Belanger2,3, Roger Nilsen2] and Brant C. Faircloth4
Address1: Department of Environmental Health Science, University of Georgia, Athens, GA 30602-2102, USA
Address2: Georgia Genomics Facility, University of Georgia, Athens, GA 30602-2102, USA
Address3: Department of Infectious Diseases, University of Georgia, Athens, GA 30602-2102, USA
Address4: Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095, USA
Contact: Travis C. Glenn
ContactEmail: travisg at uga dot edu
Copyright: 2010 Travis Glenn
This work is licensed under a Creative Commons Attribution License.
http://creativecommons.org/licenses/by/3.0/us/
Keywords: plus-A, ligation
After repairing the ends of the sheared fragments to make them blunt, we need to add a single Adenosine to the 3’ end of each DNA molecule. This will: 1) increase the efficiency of our subsequent ligation reactions with adaptors and 2) inhibit DNA fragments from ligating to each other during the linker ligations.
To add a single A to the 3’ end of each DNA molecule.
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Into the fragmented & blunt-ended DNA, add the following for a 50 μL reaction
Volume | Name | SKU | : --------------| :-----------------------------------------|--------------:| 3.0 μL | 10x NEB Buffer #2 | (comes w/ Klenow) 4.0 μL | dATP (0.25 mM -> 33 μM final) | NEB # N0440S 2.0 μL | dH2O | Amresco # E476-100ML 1.0 μL | Klenow Fragment 3’->5’ exo- | NEB # M0212 ---- | | 20.0 μL | DNA (from above) |
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Mix by pipetting and pulse spin
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Place in thermal cycler and run the program A-TAQ:
- 1 cycle of 37 C for 30 m => 75 C for 20 m => hold at 15 C
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[I don’t think we need to clean-up the sample here. It should be fine to use immediately for the next step.]