YAC end rescue for pRML1/pRML2 system:

Tao P. Zhong, Ph.D., Cardiovascular Research Center, Massachusetts General Hospital 149 13th street, Charlestown, MA 02129;
Department of Medicine, Harvard Medical School, Boston, MA 02115.

1. Digest 2 µg of yeast genomic DNA including YAC completely with SpeI or BamH1.

2. Purify DNA with Qiagen nucleotide removal columns.

3. Self-ligate the whole DNA prep with 4 µl of ligase (400u/µl, NEB) in a total volume of 200 µl at 15 ° C for 16 hours.

4. Concentrate the whole ligation product to 20 µl.

5. Transform 100 µl of DH5-alpha competent cells with 10 µl ligation product and plate on an Amp plate.

6. Pick 10 colonies from mini prep. Digest mini prep DNA with EcoR1/SpeI or EcoR1/BamH1 to release the YAC end inserts from YAC vectors. Based on the vector size of pRML1 (9.7Kb)and pRML2 (5.1Kb), both YAC ends could be rescued and distinguished.

7. The YAC end insert DNA of pRML1-based mini-YAC clone can be directly sequenced from one orientation using T3 primer.

8. The YAC end insert DNA of pRML2-based mini-YAC clone can be directly sequenced from both orientation using T7 primer and m13 reverse primer.

If you have any questions about this protocol, or the YAC library in general, please contact Dr. Tao P. Zhong (617-726-4322, zhong@cvrc.mgh.harvard.edu).

Ref: Zhong, Tao P., et al(1998) Genomics 48:136-138.