Platinum taq dna polymerase high fidelity kit

Total RNA was isolated from rat lung or rat PMVECs using RNeasy mini kit (Qiagen). After cDNA generation using iScript cDNA synthesis kit (Bio-Rad) with 1 μg RNA in a total volume of 20 μl, rat Tau cDNAs were amplified using Platinum Taq DNA polymerase high fidelity kit (Invitrogen) and rat Tau-specific primers. The primers used for PCR were designed to include restriction enzyme sites for cloning purpose and to remove the stop codon to express a tagged Tau. Forward primer: TAA GCA AGC TT(HindIII)T GAA GCA GCA TGG CTG AAC C and reverse primer: TGC TTA TCT AGA (XbaI) CAA ACC CTG CTT GGC CAA AGA G. The samples were incubated at 95 °C for 3 min, followed by 35 cycles of 95 °C 30 s, 60 °C for 30 s, and 72 °C for 60 s. A final extension step was performed at 72 °C for 5 min. Amplified PCR products were purified, double-digested with HindIII and XbaI, and cloned into the mammalian cell expression vector, pcDNA3.1-V5/His. The cloned rat tau cDNA sequences were verified by bidirectional Sanger sequencing (MCLab). The sequences have been deposited in GenBank (Accession number: 0N4R MZ604975, 1N4R MZ604976, 2N4R MZ604977, and big tau MZ604978).

For genome regions that could not be finished computationally, PCR and capillary sequencing was used. Primer sequences and annealing temperatures used for PCR and sequencing are listed in Table SA1 in the supplemental material. PCR was performed using a Platinum Taq DNA polymerase high-fidelity kit (Invitrogen), and cycling conditions were as follows: 95°C for 3 min, 30 cycles of 94°C for 20 s, the specific primer annealing temperature (see Table SA1) for 30 s, and 68°C for 3 min, and a final extension at 68°C for 7 min. PCR with primers U005 and U006 was performed using the KAPA 2G Robust PCR kit with deoxynucleoside triphosphate s (dNTPs) (Kapa Biosystems) using GC-rich buffer and the following cycling conditions: 98°C for 3 min, 25 cycles of 98°C for 30 s, the specific primer annealing temperature for 30 s, and 72°C for 2 min, and a final extension of 72°C for 2 min. PCR products were sequenced on an ABI 3730 capillary sequencer. The same primers were used for sequencing except where noted. PCR products were merged into the de novo genome assembly using Gap5 software (31 (link)) to generate a final genome sequence for each strain. Regions where PCR failed or which could not be fully resolved were filled with Ns.

Naive mature B cells, which were sorted from peripheral blood mononuclear cells of the patient, were used for total RNA preparation with the RNeasy Plus Kit (Qiagen). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PAX5 full-length transcripts were amplified by PCR using the Platinum Taq DNA Polymerase High Fidelity kit (Invitrogen) with the primers shown in Table S7 and were cloned into the pcDNA3.1(+) expression vector. Plasmid DNA of single clones was prepared, followed by Sanger sequencing of the cloned PAX5 cDNA sequences.