Confirmation of ccpN point mutation and ccpN in-frame deletion by PCR.

The PCR product was analyzed by agarose gel electrophoresis. A 1(Fermentas) was used as a molecular weight marker (lane M). A. Confirmation of the ccpN point mutation. Fragments were amplified using ccpN-Mut-P7/ccpN-Mut-P8 as primers. Each lane showed amplified DNA generated from a DNA template: lane 1, BUK-1C (ΔccpN::upp-cassette); lane 2, BUK-1 (ccpN-mut-Ala 130 to Ser); lane 3, dsDNA PCR fragment (positive control); lane 4, BUK (ccpN-wild type) (negative control). B. Confirmation of the ccpN in-frame deletion. Fragments were amplified using ccpN-Del-P1/ccpN-Del-P6 as primers. Each lane showed amplified DNA generated from a DNA template: lane 5, BUK-2C (ΔccpN::upp-cassette); lane 6, BUK-2 (ΔccpN); lane 7, dsDNA PCR fragment (positive control); lane 8, BUK (ccpN-wild type) (negative control).