Mini preparation kit

Blood was collected from patients before surgery. DNA from blood was isolated with Qiagen mini-preparation kits and genotyped for the two polymorphisms in the MTHFR gene (C677T and A1298C). Genotyping was performed using KASPar chemistry, a competitive allele-specific PCR genotyping system (http://www.lgcgenomics.com) according to manufacturer’s instructions. The PCR was carried out in plates of 96 wells, in a total reaction volume of 8 μl using 10 ng of genomic DNA, 2 μl 2X KASPar reaction mix and 0.11 μl of the assay mix. The PCR conditions were: an initial denaturation at 94°C for 15 minutes, 10 touch-down cycles of 20 s at 94°C and 60 s at 61-55°C (temperature decrement by 0.6°C per cycle) and additional 25–30 cycles at 20 s at 94°C and 60 s at 55°C. Genotypes in amplified products were determined by differences in VIC and FAM fluorescent level in plate read operation on ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) using SDS 2.2 Software. Post operation data were transferred to Microsoft Excel files and converted into genotype information. The genotype quality control was validated through DNA sequencing in 5% of the samples.

Synthetic gene blocks were assembled into pTYB1 vector (NdeI and XhoI digested) using a Gibson assembly kit (NEB) and the DNA inserts were selected by plasmid DNA transformation and the correct insert was verified by DNA sequencing. Plasmids were prepared by plasmid mini-preparation kits from Qiagen or Sigma. Gene blocks encoding Pam7902I catalytic mutants were cloned into pTYB1 expression vector and the inserts were verified by DNA sequencing to contain the desired mutations.

The positive colony was picked and streaked into the fresh LB agar plate with kanamycin as a resistant marker. Once the desired growth was attained, a single colony was inoculated into the 5 ml of LB broth containing appropriate antibiotic and incubated 37°C for 12-16 hrs. The construct was extracted by using a mini plasmid extraction procedure (Qiagen mini preparation kit) as described above. The restriction analysis was carried out as follows, 5 ul of universal fast digest buffer (33mm Tris Acetate, 10 mM Magnesium Acetate, 66mM Potassium Acetate and 0.1 mg/ml BSA), 1 µl of Hind III and 1 µl of EcoRI, 1µl of BamHI, 1 µl of XhoI, 10 µl of construct (hflc + pET-28a, hflk + pET-28a, yhc1 + pET-28a) and 33 µl of Milli-Q. The digestion was carried out at 37°C for 1 hour. Agarose gel electrophoresis was carried out with empty pEt-28a, construct single and double digest and finally amplified product of hflc, hflk, and yhc1 in ascending order with molecular markers on both sides of the gel.