Platinumr taq dna polymerase

Total RNA was prepared using the PureLink Micro‐to Midi Total RNA Purification System (Invitrogen). RT‐PCR was performed using the SuperScript III One‐Step RT‐PCR System with Platinum^R Taq DNA Polymerase (Invitrogen) to analyze the expression of pluripotency markers. The following conditions were used: 55 °C for 30 min for reverse transcription, 94 °C for 2 min to inactivate the reverse transcriptase and activate the polymerase, 40 cycles at 94 °C for 15 s, 55 °C for 30 s, 68 °C for 1 min, and 68 °C for 5 min for the final extension. The following products were analyzed: OCT4 (315 bp), NANOG (285 bp), and GAPDH (513 bp). The primers were as follows: OCT4, 5′‐GAA GGT ATT CAG CCA AAC GAC‐3′ (forward) and 5′‐GTT ACA GAA CCA CAC TCG GA‐3′ (reverse); NANOG, 5′‐TGC AAA TGT CTT CTG CTG AGA T‐3′ (forward) and 5′‐GTT CAG GAT GTT GGA GAG TTC‐3′ (reverse); GAPDH, 5′‐GTC CAT GCC ATC ACT GCC A‐3′ (forward) and 5′‐TTA CTC CTT GGA GGC CAT G‐3′ (reverse) [8, 9].

Genomic DNA was extracted from the peripheral blood leukocytes of the patients and their parents. CDKL5 gene (NM_003159.2) mutations were analyzed by polymerase chain reaction (PCR) and direct sequencing. PCR was carried out in 25 ul reaction system with 2 × GC Buffer I 12.5 ul, deionized H₂O 4.5 ul, 5′ primer 2 ul (5Pmol), 3′ primer 2 ul (5Pmol), dNTPs 2ul, platinum rTaq DNA polymerase (Invitrogen) 1ul and DNA template 1 ul. The annealing temperature was 59°C for exon1-21 except for exon 5 with 57°C. Primer sequences and polymerase chain reaction conditions are available upon request. GenBank accession number NM_003159.2 was used as the DNA reference sequence. The nomenclature used follows the HGVS mutation nomenclature guidelines (http://www.hgvs.org). Pathogenicity prediction of previously unreported variants was performed using Alamut version2.0 (Interactive Biosoftware, Rouen, France). When no mutation was founded, MLPA (SALSA MLPA kit P189 CDKL5, MRC-Holland, Amsterdam, Holland) was performed to detect large deletions or duplications of CDKL5 gene, according to the previous reports [16 (link),17 (link)].

Total RNA was isolated from mouse brain and pancreatic acini using the RNAeasy Midi kit (Qiagen, Valencia, CA) and cDNA was synthesized using the iScript^TM cDNA Synthesis kit (Bio-Rad Laboratories, Inc, USA) using random hexamers. PCR was carried out using 2μl first-strand cDNA in a 50μl reaction volume containing [1× PCR buffer–Mg, 1.5 mM MgCl₂, dNTP mix (0.2 mM each dNTP), 0.2 μM each primer (forward and reverse), and 2U/rxn of Platinum^RTaq DNA polymerase (Invitrogen, Carlsbad, CA)]. The specifics for both mouse and human primers and amplification conditions are as follows. Mouse primers used were based on those previously used in rat as the sequence in question is the same, α7nAChR F: 5’-ATCTGGGCATTGCCAGTATC-3’, R: 5’-TCCCATGAGATCCCATTCTC-3’ [1 (link), 16 (link)]. Amplification conditions were initial denaturation (3 min, 94°C) then 45 cycles of denaturation (94°C, 45 sec), annealing (49°C, 30 sec), and extension (72°C, 30 sec). For human PCR the above primers were modified to correspond to the human sequence α7nAChR F: 5’-TTCTGGGCATTGCCAGTACC-3’, R: 5’-TCCCACAGGTCCCATTCTC and the amplification conditions used were the same as used above for mouse except that the annealing step which was modified to (51°C, 30 sec). PCR products were analyzed on 1× TAE agarose gel that contained ethidium bromide.