Pcr buffer

Genotyping was performed by amplification refractory mutation system PCR (ARMS-PCR). The IL-12p40 promoter region was screened for two different already described alleles (GC/(GC) _del(CTC TAA)_ins) (rs17860508 [5 (link)]). The longer allele IL-12p40 pro1 is detected by the specific primer IL-12p40 5–8 (TGT CTC CGA GAG AGG CTC TAA), the 4 bp shorter allele IL-12p40 pro2 by IL-12p40 5–10 (TGT CTC CGA GAG AGG GCT GT). As generic 3′ primer IL12p40 3–5 (TGG AGG AAG TGG TTC TCG TAC) was used for both reactions (Primers from Eurogentec, Cologne, Germany). As control primers derived from the C reactive protein-gene CRP 3 and 5 were employed (CRP 3: CCA GCC TCT CTC ATG CTT TGG TTG GCC AGA CAG, CRP5: GGG TCG AGG ACA GTT CCG TGT AGA AGT GGA).
The reaction mix contained 10–20 ng genomic DNA, 1x PCR Buffer (Qiagen, Hilden, Germany, containing 15 mM MgCl₂), 1.5 μl MgCl₂ (25 mM), 0.5 μl dNTP 10 mM, 1U Top Taq DNA Polymerase (Qiagen Hilden, Germany), 5 pmol of each IL-12p40 primers and 2.5 pmol of each CRP primer. Taq activation was performed by an initial step of 95 °C (15 min), followed by 35 cycles (94 °C for 30s, 65 °C for 30s, 72 °C for 30s). PCR product was visualized in a 2.5 % agarose gel with 0.01 % ethidium bromide.

RT-PCR was carried out in 200 μl tubes (Bioplastics, Landgraaf, The Netherlands) using 1 μl of cDNA as template in 25 μl PCR mixture containing final concentrations of 2 mM MgCl₂, 200 μM of each NTP, 0.5 μM of each primer and 0.625 units Taq DNA polymerase (HotStarTaq, Qiagen) in 1xPCR buffer (Qiagen). Initial denaturation was performed at 95°C for 15 min, followed by 40 cycles of 15 sec at 94°C, 30 sec at the primer specific annealing temperature (Table 1), and 45 sec at 72°C. Final extension was performed at 72°C for 10 min. The PCR products were resolved by electrophoresis in 1% agarose gels containing ethidium bromide. A 100 base pair (bp) DNA ladder (Invitrogen) was included as a reference for fragment size.

To observe methylation levels of Alu in samples, the sodium-bisulfite-treated DNA in each sample was amplified by PCR containing 1x PCR buffer (Qiagen, Germany), 0.2 mM of deoxynucleotide triphosphate (Promega, USA), 1 mM of magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and 0.3 μM primer pairs: ALU-BRev (5′-CTAACTTTTTATATTTTTAATAAAAACRAAATTTCAC CA-3′) where R = A and G and Y = C and T. For Alu amplification, the program was set as follows: 95 °C for 15 min, 40 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, followed by a final extension of 72 °C for 7 min [20 (link)]. Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight. The cut PCR products were analyzed by 8% acrylamide gel and SYBR stain (Lonza, USA). The band intensity of Alu methylation was observed and measured by typhoon fla 7000 and ImageQuanNT Software (Amersham biosciences, UK) (Additional file 1: Figure S1) [11 ].