Isolation of Specific DNA Clones Using Biotinylated dD-Loops
|
|
Colonies (presep) |
Colonies (postsep) |
||||
|---|---|---|---|---|---|---|
| Kan/Tet |
Kan |
Tet |
Kan |
Tet |
||
| 1:0 | TNTC | 0 | >4000 | 0 | ||
| 1:10 | TNTC | TNTC | 2543 | 0 | ||
| 1:100 | 2060 | TNTC | 282 | 0 | ||
| 1:1,000 | 1132 | TNTC | 30 | 0 | ||
| 1:10,000 | 280 | TNTC | 2 | 0 | ||
| 1:100,000 | 17 | TNTC | 1 | 0 | ||
| 0:1 | 0 | TNTC | 0 | 0 | ||
| 1:10 (no RecA)
|
TNTC
|
TNTC
|
0
|
0
|
||
-
Shown in the table are results from a typical sequence capture experiment. Kanamycin homologous oligonucleotides were tested to see if they could separate kanamycin-resistant pWE15 plasmids from tetracycline-resistant pBR322 plasmids by sequence-specific interaction. The first column shows the ratio of pWE15 plasmids to pBR322 plasmids in the experimental target mix. In the following columns, we present results before separation (Colonies presep) and after the dD-Loop complexes labeled with biotin were separated by binding to strepavidin-conjugated paramagnetic beads (Colonies postsep). The numbers presented are actual colony numbers obtained after transforming equal samples of each condition into bacteria and replica plating for kanamycin (Kan) and tetracycline (Tet) LB plates. The process was routinely sensitive enough to isolate specific clones represented as little as 1 in 105 clones in a gene library.
