Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Table 1 Testing of USER Friendly cloning for single step four fragment cloning

			No. of correct colonies/total no. of colonies			Average
Vector	Insert 1	Insert 2	1.	2.	3.
pRF-HU	PKS1-A1/A2	na	87/91	100/103	114/120	104.7 (95.9%)
pRF-HUE	PKS1-O1/O2	na	59/61	60/63	66/70	64.7 (95.4%)
pRF-HU2	PKS1-A1/A2	PKS1-A3/A4	47/53	52/61	45/55	56.3 (85.2%)
pRF-HU2E	PKS1-O1/O2	PKS1-O3/O4	38/48	39/45	40/49	47.3 (82.4%)

USER cloning: Results from three independent replicates where one or two PCR amplicons are cloned into the designed USER vectors. All transformants were first tested for the presence of the desired insert and then for the orientation of the insert in the vector. All transformants that were positive for inserts also had the correct orientation. The negative control reactions, where no PCR amplicons were added to the reactions, resulted in zero colonies.

ISSN: 1471-2199