Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage
Abstract
:1. Introduction
2. Results
2.1. Cell Cultures and Characteristic Tests
2.2. Vector Preparation and High-Molecular-Weight (HMW) Genomic DNA Preparation
2.3. Characterization and Insert Size Testing of BAC Library
2.4. The Testing of Library Stability
2.5. BAC End Sequence Analysis
2.6. BAC Library Screening
3. Discussion
4. Materials and Methods
4.1. Preparation of BAC Vector
4.2. High-Molecular-Weight (HMW) Genomic DNA Preparation
4.3. Ligation and Transformation
4.4. Large-Scale BAC Clone Production
4.5. Insert Size Distribution of Siberian Tiger BAC Library
4.6. Library Pooling and BAC Library Screening
4.7. BAC End Sequencing
5. Conclusions
Acknowledgments
Conflicts of Interest
References
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Sub-library | Sample | Vector | Bacterial strain | Cloning enzyme | Total clones | Plate No. | Non-insert clone (%) | Average insert size a (kb) |
---|---|---|---|---|---|---|---|---|
BAC11-B | Blood cells | pBeloBAC11 | E. coli DH10B | HindIII | 92,160 | 1–240 | 2.1 (2) | 126.5 |
pCC1-B | Blood cells | Copy Control™ pCC1 BAC™ | EPI300™-T1R E. coli | HindIII | 23,040 | 241–300 | 5.2 (5) | 116.7 |
BAC11-F | Fibroblasts | pBeloBAC11 | E. coli DH10B | HindIII | 23,040 | 301–360 | 3.1 (3) | 114.3 |
pCC1-F | Fibroblasts | Copy Control™ pCC1 BAC™ | EPI300™-T1R E. coli | HindIII | 15,360 | 361–400 | 1.0 (1) | 110.1 |
Total | 153,600 | 400 | 2.6 | 116.5 |
Marker name | Location Chr | Number of positive superpools | Number of positive clones |
---|---|---|---|
KF463190 | A1 | 4/2 | 6/2 |
KF463196 | A2 | 3/1 | 4/1 |
KF463200 | A3 | 3/0 | 9/3 |
KF463203 | B1 | 2/0 | 5/2 |
KF463210 | B2 | 1/2 | 4/2 |
FCA201 | B3 | 3/2 | 4/3 |
FCA069 | B4 | 3/1 | 4/1 |
KF463223 | C1 | 3/NF | 6/0 |
FCA043 | C2 | 3/1 | 5/2 |
KF463232 | D1 | 4/1 | 6/1 |
KF463233 | D2 | 2/2 | 4/4 |
KF463238 | D3 | 2/3 | 5/3 |
KF463238 | D4 | NF/2 | 0/2 |
KF463245 | E1 | 4/1 | 8/1 |
KF463247 | E2 | 3/1 | 5/2 |
KF463252 | E3 | 3/2 | 5/2 |
KF463255 | F1 | 2/NF | 4/1 |
FCA094 | F2 | 4/2 | 5/1 |
KF463257 | X | 2/1 | 3/1 |
SRY | Y | 1/0 | 1/0 |
Average number of positive clone | 4.90/1.84 |
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Liu, C.; Bai, C.; Guo, Y.; Liu, D.; Lu, T.; Li, X.; Ma, J.; Ma, Y.; Guan, W. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage. Int. J. Mol. Sci. 2014, 15, 4189-4200. https://doi.org/10.3390/ijms15034189
Liu C, Bai C, Guo Y, Liu D, Lu T, Li X, Ma J, Ma Y, Guan W. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage. International Journal of Molecular Sciences. 2014; 15(3):4189-4200. https://doi.org/10.3390/ijms15034189
Chicago/Turabian StyleLiu, Changqing, Chunyu Bai, Yu Guo, Dan Liu, Taofeng Lu, Xiangchen Li, Jianzhang Ma, Yuehui Ma, and Weijun Guan. 2014. "Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage" International Journal of Molecular Sciences 15, no. 3: 4189-4200. https://doi.org/10.3390/ijms15034189