The following retroviral vectors encoding replication-deficient retroviruses were used: pMSCV-IRES-GFP (MIG) and pMSCV-GFP-IRES-hN-RasG12D (NRAS) were previously described (20 (link)). We obtained a shuttle vector containing constitutively active form of MEK [caMEK, MEK1-S218E/S222D (31 (link))] from Addgene (plasmid #40809). The caMEK gene cassette was amplified from this plasmid using the primers caMEK-NotI (5′-AAAGCGGCCGCGTTACCCGGGTCCAAAA-3′) and caMEK-SalI (5′-AATGTCGACTTAGACGCCAGCAGCATG-3′) and AccuTaq polymerase (Sigma). The PCR product was cloned between NotI and SalI in the retroviral pMSCV-IRES-GFP vector (20 (link)) to generate the pMSCV-caMEK-IRES-GFP plasmid. These vectors are based on the Murine Stem Cell Virus (MSCV) retroviral expression system and contain an internal ribosome entry site (IRES) for bicistronic gene expression. We obtained the ERK2 shRNA and luciferase shRNA sequences in shuttle vectors from Open Biosystems. The shRNA sequences were isolated from these vectors as XhoI-EcoRI fragments that were then cloned into the MSCV-LTRmiR30-PIG (LMP) vector (Open Biosystems), kindly donated by John Cambier (University of Colorado), to create the MSCV-LMP-ERK2-shRNA and MSCV-LMP-luc-shRNA plasmids. Retroviral particles were produced as described previously (20 (link)).
Accutaq polymerase
Manufactured by Merck Group
AccuTaq polymerase is a thermostable DNA polymerase used for PCR amplification. It exhibits high fidelity and robust performance in a variety of PCR applications.
Automatically generated - may contain errors
4 protocols using accutaq polymerase
1
Generating Retroviral Vectors with Constitutively Active MEK
2
Genetic Analysis of Core GGE Phenotypes
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Twenty patients with core GGE phenotypes, including some with febrile seizures, were recruited. All patients and relatives or their legal representatives gave written informed consent to participate in this study. Ethical approval was obtained from the responsible local authorities. The clinical information for the two families in which the mutations have been detected is presented in the Appendix S1 .
Genomic DNA was extracted from peripheral blood leukocytes using a salting‐out method. Polymerase chain reaction (PCR) was performed with 50‐ng genomic DNA, 10 pmol of each primer, 200 μm dNTP, 50 mm Tris‐HCl, 15 mm ammoniumsulfate, 2.5 mm MgCl2, 5% dimethyl sulfoxide (DMSO), 0.75 U AccuTaq Polymerase (Sigma‐Aldrich) in a total volume of 25 μl. Primers were designed to amplify the entire coding region and adjacent intron sequences of the candidate genes. PCR was performed in an MJ Research thermocycler with the following conditions: 35 cycles of denaturation at 95°C for 30 s, annealing temperatures ranging from 62°C to 90°C for 30 s, and extension at 68°C degree for 90 s. The amplicons were purified and subsequently sequenced.
For all missense variants, we assessed genotype and allele frequencies in a total of 230 ethnically matched controls using specific restriction digestion assays. All available members of the families were genotyped for the cosegregation analysis.
Genomic DNA was extracted from peripheral blood leukocytes using a salting‐out method. Polymerase chain reaction (PCR) was performed with 50‐ng genomic DNA, 10 pmol of each primer, 200 μ
For all missense variants, we assessed genotype and allele frequencies in a total of 230 ethnically matched controls using specific restriction digestion assays. All available members of the families were genotyped for the cosegregation analysis.
3
Validating Alternative Splicing Events in Erythroblasts
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Many alternative splicing events were verified by manual inspection of mapped reads on the human genome browser, to filter out exons not supported by multiple exon-inclusion and exon-skipping reads. In selected cases, RT-PCR analysis was performed to visualize the amplified products corresponding to inclusion and skipping events. Total RNA was reverse-transcribed into cDNA using random primers and the Superscript III First Strand Synthesis System (Invitrogen), and PCR analysis was performed using AccuTaq polymerase (Sigma) using primers located in constitutive exons located upstream and downstream of each candidate alternative exon. Primer sequences are given in Supplementary Table S4 . For initial RT-PCR validations, RNA from unsorted day 9 (proE-enriched) and day 16 (orthoE-enriched) cells was used; these results were subsequently confirmed with RNA purified from highly enriched (FACS-sorted) proEs and orthochromatophilic erythroblasts.
4
Gene Expression Analysis by RT-PCR
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Total RNA was extracted from cells using RNeasy Mini kit (Qiagen, Germany). The concentrations of mRNA were determined spectrophotometrically, and ~200 ng of total mRNA was used for reverse transcription. Single-stranded cDNA was obtained using Enhanced AMV Reverse Transcriptase (Sigma) and primed with Anchored Oligo (dT) 23 . Subsequently, the RT-PCR product was subjected to PCR using Taq polymerase using specific primers listed in Table 1. The following conditions were applied for PCR. PCR was carried out using 5 μl of the RT products and Accu Taq polymerase (Sigma) in a final volume of 50 μl. The samples were denatured at 95 °C for 30 s, annealed at 56 or 70 °C for 30 s and extended at 72 °C for 1 min. The cycle of incubations was repeated 40 times. The PCR products were identified after electrophoresis in 1.2% agarose gels, and the obtained products were verified by DNA sequencing using ABI Prism 310 DNA Analyser (Applied Biosystems).
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