Table 2.
Summary of LR Reactions of Two-Segment Cloning
|
LR reaction (10 μL) |
DNA topology |
Input DNA (ng) |
Insert size (bp) |
DNA size (bp) |
fmoles |
AmpR cfua 2 h |
AmpR cfu 6 h |
AmpR cfu 24 h |
|---|---|---|---|---|---|---|---|---|
| L4-NLS-R1.1 | ccc | 40 | 27 | 2673 | 23 | 44 (4/4)b | 106 (4/4) | 97 (4/4) |
| L1.1-eGFP-L2.1 | ccc | 40 | 719 | 3261 | 19 | |||
| pDESTCMV42 (R4-R2.1) | linear | 134 | 7302 | 27 | ||||
| L4-Transferrin-R1.1 | ccc | 30 | 155 | 2802 | 16 | 125 (4/4) | 900 (4/4) | 1675 (4/4) |
| L1.1-eGFP-L2.1 | ccc | 40 | 719 | 3261 | 19 | |||
| pDESTCMV42 | linear | 134 | 7302 | 27 | ||||
| L1.1-Transferrin-L2.1 | ccc | 17 | 155 | 2697 | 9 | 2588 (4/4) | 2425 (4/4) | 2750 (4/4) |
| R2.1-no ATG eGFP-L3 | ccc | 67 | 713 | 3356 | 31 | |||
| pDESTCMV13 (R1.1-R3) | linear | 134 | 7302 | 27 | ||||
| L4-SDHD-R1.1 | ccc | 40 | 454 | 3001 | 20 | 462 (4/4) | 438 (4/4) | 625 (4/4) |
| L1.1-eGFP-L2.1 | ccc | 40 | 719 | 3261 | 19 | |||
| pDESTCMV42 (R4-R2.1) | linear | 134 | 7302 | 27 | ||||
| L1.1-SDHD-L2.1 | ccc | 100 | 454 | 2896 | 52 | 925 (4/4) | 675 (4/4) | 862 (4/4) |
| R2.1-no ATG eGFP-L3 | ccc | 67 | 713 | 3356 | 31 | |||
| pDESTCMV13 (R1.1-R3) | linear | 134 | 7302 | 27 | ||||
| L1.1-β-arrestin-L2.1 | ccc | 100 | 1229 | 3765 | 40 | 2667 (4/4) | 6222 (4/4) | 7111 (4/4) |
| R2.1-no ATG eGFP-L3 | ccc | 100 | 713 | 3356 | 45 | |||
| pDESTCMV13 (R1.1-R3) | linear | 200 | 7302 | 42 | ||||
| No Clonase | ||||||||
| L4-NLS-R1.1 | ccc | 40 | 27 | 2673 | 23 | 12 | ||
| L1.1-eGFP-L2.1 | ccc | 40 | 719 | 3261 | 19 | |||
| pDESTCMV42 (R4-R2.1) | linear | 134 | 7302 | 27 | ||||
| No Clonase | ||||||||
| L1.1-β-arrestin-L2.1 | ccc | 100 | 1229 | 3765 | 40 | 0 | ||
| R2.1-no ATG eGFP-L3 | ccc | 100 | 713 | 3356 | 45 | |||
| pDESTCMV13 (R1.1-R3)
|
linear
|
200
|
|
7302
|
42
|
|
|
|
-
(ccc) Supercoiled, (Amp) ampicillin resistant.
-
↵a Colony forming units per milliliter transformation obtained from transformation of 1 μL BP reaction into 50 μL competent DH5α cells (Invitrogen) 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. Cotransforming Entry Clone DNA was observed in 6 of 48 samples.
