Bacterial isolates were grown overnight on Columbia blood agar plates. Three colonies of bacterial cells were suspended in 20 μl lysis buffer (0.25% SDS, 0.05 N NaOH) and incubated at 95˚C for 5 min. The cell lysate was spin by short centrifugation and diluted with 180 μl buffer (10 mM Tris-DCl, pH 8.5). After
thoroughly mixing, another centrfugation for 5 min at 16,000 x g was performed to remove cell debris. Supernatants were frozen at -20˚C until further use. Two and a half μl of the lysate was used in the PCR reaction.
From isolates that did not yield a PCR product, chromosomal DNA was extracted using the QiaAmp Blood kit (Qiagen inc., Valencia, CA, USA) according to the manufacturers instruction for Gram-positive bacteria with some minor changes in the lyses of the bacteria. From an overnight culture 1.5 ml was spin for 2 min, suspended in 200 µl of 10 mM Tris-1 mM EDTA pH8.0 and 10 µl of a 50-mg/ml solution of egg white lysozym (Roche) and incubated at 37˚C for 15 min. The bacteria were lysed by the addition of 30 µl of 10% sodium dodecyl sulfate and 20 µl of a 20-mg/ml proteinase K (Merck) solution and incubated at 65˚C for 1 hour. Subsequently, the protocol according to the manufacturers instructions was used.
The PCR conditions were not the same for all of the amplification reactions. In all cases the initial denaturation was at 95˚C for 15 min and a final extension of 5 min at 72˚C.
Alterations: The extention times are increased, the VNTR 8 and 9 PCR programs are now the same as VNTR 7 and 10. After the TD part, the remaining cycles increased from 25 to 30 cycles.
- For VNTR-1, 35 cycles of 30 s at 94˚C, 30 s at 52˚C and 1 min at 72˚C were performed.
- For VNTRs 2 a Touch Down (TD) PCR was developed that included 10 cycles of 30 s 94˚C, 30 s 70˚C (see below) and 4 min 72˚C. The annealing temperature during the first cycle was 70˚C and decreased 1˚C at each cycle during the next 9 cycles. During the remaining 30 cycles, an annealing temperature of 60˚C was used.
Note: For some strains it is difficult to obtain a PCR product for VNTR-2. When we used Ready-to-go beads from Amersham instead of HotStarMastermix, in most cases a positive result was obtained.
- For VNTRs 7, 8, 9 and 10 the initial annealing temperature was 65˚C and decreased to 55˚C. Extention is 1 min at 72˚C. Reactions were performed in 25 µl volumes with HotStar Taq polymerase and HotStar Master Mix buffers from Qiagen (Qiagen inc.).