Table 3.
Defined Replacement of att-Flanked Segments With a BP Reaction
|
BP reaction (10μL) |
DNA topology |
Input DNA (ng) |
DNA size (bp) |
fmoles |
AmpR CmR cfu 20 ha |
|---|---|---|---|---|---|
| pEXP425-[B4-STE2 promoter-B1.1-STE2-B2.1] | ccc | 300 | 8925 | 50 | 3206 |
| pDONR221 (P1-ccdB CmR-P2) | ccc | 75 | 4759 | 24 | (1/6)b |
| No Clonase | 928 | ||||
| pEXP425-[B4-STE2 promoter-B1.1-STE2-B2.1] | ccc | 300 | 8925 | 50 | 55 |
| pDONR221 (P1-ccdB CmR-P2) | linear | 75 | 4759 | 24 | (4/6) |
| No Clonase | 0 | ||||
| pEXP425-[B4-STE2 promoter-B1.1-STE2-B2.1] | linear | 300 | 8925 | 50 | 562 |
| pDONR221 (P1-ccdB CmR-P2) | ccc | 75 | 4759 | 24 | (5/6) |
| No Clonase | 8 | ||||
| pEXP425-[B4-STE2 promoter-B1.1-STE2-B2.1] | linear | 300 | 8925 | 50 | 94 |
| pDONR221 (P1-ccdB CmR-P2) | linear | 75 | 4759 | 24 | (6/6) |
| No Clonase
|
|
|
|
|
0
|
-
↵a Colony forming units per milliliter transformation obtained from transformation of 1 μL reaction into 50 μL competent DB3.1 cells at 1 × 108 cfu/μg pUC.
-
↵b Number of correct clones vs. total number of clones analyzed based on restriction enzyme analysis of miniprep DNA. (ccc) Supercoiled; (AmpR) ampicillin resistant; (CmR) chloramphenicol resistant.
