The constructs for overexpression, namely pEA-Flag-BmSiwi and pEA-BmAgo3-MycHis, as well as pEA-pac, which contains the ORF of puromycin resistance gene (negative control), were obtained according to the methodology described by Kolliopoulou and Swevers [25 (link)]. In short, to generate the Bm-Siwi expression construct, the complete ORF was amplified by PCR (Platinum Taq HF DNA polymerase; Invitrogen, Waltham, MA, USA). The forward primer contained a BglII-site for cloning in-frame with the N-terminal Flag tag of a modified pEA lepidopteran expression vector [26 (link)], as well as a Kozak initiation sequence [27 (link)]. Similarly, in order to create an expression construct for Bm-Ago3, the ORF was cloned in the pEA-MycHis lepidopteran expression vector [26 (link)] after amplification by PCR (Platinum Taq HF DNA polymerase; Invitrogen, Waltham, MA, USA). Both the forward and reverse primers contained a BglII cloning site, while the forward primer contained a Kozak initiation sequence [27 (link)] and an ATG start codon. The reverse primer was appropriately designed for in-frame cloning with the C-terminal MycHis tag of the pEA-MycHis vector. Both pEA-Flag-BmSiwi and pEA-BmAgo3-MycHis were verified by sequencing. The primer sequences are indicated in Table S1 .
Platinum taq dna polymerase hf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Platinum Taq DNA Polymerase HF is a high-fidelity DNA polymerase used for DNA amplification in various molecular biology applications. It possesses 3'→5' exonuclease proofreading activity, which ensures accurate DNA synthesis with reduced error rate compared to standard Taq DNA polymerase.
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3 protocols using platinum taq dna polymerase hf
1
Overexpression Constructs for BmSiwi and BmAgo3
2
Zebrafish Progranulin Paralogs Rescue Assay
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The coding sequence of zebrafish (zf) pgrn‐a (NM_001001949.2; in pSPORT1 Vector) and the pgrn‐a paralog, zf pgrn‐b (NM_212738.1; in pBK‐CMV Image Clone from Open Biosystems, GE Dharmacon, Lafayette, CO), were PCR amplified starting from the ATG (primer sequences listed in Supporting Information Table S4), therefore removing the 5′UTR MO recognition sequence, using Platinum Taq DNA Polymerase HF (Invitrogen, Carlsbad, CA). cDNAs were subcloned into the pGEM‐T Easy Vector (Promega, Madison, WI) using standard protocols, linearized with SacI‐HF (New England Biolabs, Ipswich, MA), and capped RNAs were transcribed using the mMESSAGE mMACHINE T7 kit (Ambion, Thermo Fisher Scientific, Halethorp, MD). Human progranulin (hGRN; GE Healthcare clone ID 3457813 in pCMV‐SPORT6) and eGFP (pCS2+‐EGFP) were linearized with NotI‐HF and transcribed with SP6. For rescue experiments, zf pgrn‐a (25 pg/embryo), zf pgrn‐b (25 pg/embryo), or human progranulin (hGRN; 400 pg/embryo) was coinjected with 5′UTR MO (.25 ng/embryo) at the one cell stage. As a negative control, eGFP mRNA (25 pg/embryo) was coinjected with the 5′UTR MO (.25 ng/embryo). At 72 hpf Embryos were treated with EdU, sacrificed, and processed for IHC.
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Zebrafish Progranulin Paralogs: Functional Assessment
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