A total of 100 ng total RNA was used to generate cDNA in a 20 µl reaction using a qScript mastermix (QuantaBio, MA, USA) that contained random hexamers. Reverse transcription was performed in a PCR machine with the following parameters: 22 °C for 5 min, 42 °C for 30 min, 85 °C for 5 min and 4 °C hold. For a non-RT control, reactions were set up in duplicate but without RT enzyme. The cDNAs were diluted 1 : 10 and 2 µl of each was used for subsequent PCR reactions with one unit of Taq polymerase (New England Biolabs, MA USA), 200 uM dNTPs (New England Biolabs, MA, USA) and 1× standard Taq polymerase buffer in a 25 µl reaction. The primers used for RT-PCR analysis of cas genes are listed in Table 1 . Following initial denaturation for 3 min at 95 °C, the PCR conditions were as follows: 20 cycles (16S control PCR) or 25 cycles (cas genes) of 95 °C for 30 s, annealing at 57 °C for 30 s and an extension at 72 °C for 30 s. A total of 5 µl of the PCR reaction was imaged by gel electrophoresis. The RT-PCR experiments were run independently twice (different bacterial cultures), and on one of these, the PCR step was performed twice on the same cDNA. The gel (Fig. 4) is from one of the independent experiments. Densitometry was not performed as the difference between the log and stationary phases was clear.
Taq polymerase
Manufactured by New England Biolabs
Sourced in United States, United Kingdom, Germany, Canada
Taq polymerase is a thermostable DNA polymerase enzyme isolated from the bacterium Thermus aquaticus. It is commonly used in the polymerase chain reaction (PCR) technique for the amplification of DNA sequences.
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Lab products found in correlation
T4 dna ligase, by New England Biolabs (11 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (11 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Thermopol buffer, by New England Biolabs (9 mentions)
Dntps, by New England Biolabs (8 mentions)
Qiaprep spin miniprep kit, by Qiagen (8 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Taq ligase, by New England Biolabs (7 mentions)
T4 ligase, by New England Biolabs (6 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (6 mentions)
Yoyo 1, by Thermo Fisher Scientific (6 mentions)
Thermopol reaction buffer, by New England Biolabs (6 mentions)
Restriction endonuclease, by New England Biolabs (5 mentions)
Pqe30, by Qiagen (5 mentions)
Irysprep reagent kit, by Bionano Genomics (5 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Random hexamer, by Thermo Fisher Scientific (5 mentions)
Taq buffer, by New England Biolabs (5 mentions)
Phusion polymerase, by New England Biolabs (4 mentions)
301 protocols using taq polymerase
1
Quantification of cas Gene Expression
2
Mitochondrial DNA Sequencing and Analysis
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We sequenced a part of the hyper-variable domain of the control region of mitochondrial DNA using the oligonucleotides 1F (5′-AATTACTTTGGTCTTGGTAAACC-3′ ) and 6R (5′-GCCCTGAAGTAAGAACCAGATG-3′ ) Horn [19] . Polymerase chain reactions (PCR) took place in a final volume of 15 µl and contained 0.1 U of Taq polymerase (New England Biolabs), 1.5 µL of 10× Taq polymerase buffer, 1.8 mM MgCl2, 0.2 mM each dNTP, 0.25 mg/ml BSA, and 0.3 µM each primer. We used the following thermal cycling parameters: 5 min at 94°C, 40 PCR cycles (55 s at 94°C, 45 s at 54°C, 45 s at 72°C) plus 10 min at 72°C. PCR products were purified using Exo-Sap-it (Affymetrix). We sequenced the amplicons on a 3730 DNA Analyzer (Applied Biosystems) in both directions with Big-Dye Terminator v3.1 chemistry (ABI), aligned the sequences with ClustalW 1.83 [20] (link) and calculated basic sequence analyses and pairwise genetic distances in MEGA 5.10 [21] (link).
A phylogenetic network was constructed in TCS 1.21 [22] (link) by statistical parsimony, treating alignment gaps as fifth state. We combined the beaver haplotypes obtained in this study with 12 additional haplotypes downloaded from GenBank (NCBI). New mtDNA sequences were deposited in GenBank (r2 = KF731635; r3 = KF731636; e = KF731637; c = KF731638).
A phylogenetic network was constructed in TCS 1.21 [22] (link) by statistical parsimony, treating alignment gaps as fifth state. We combined the beaver haplotypes obtained in this study with 12 additional haplotypes downloaded from GenBank (NCBI). New mtDNA sequences were deposited in GenBank (r2 = KF731635; r3 = KF731636; e = KF731637; c = KF731638).
3
Exon Skipping Protocol for RHO Gene
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For the exon skipping experiments using the minigene splice vectors, the target region was amplified from the synthesized cDNA using Taq polymerase (M0491L, New England Biolabs, Ipswich, MA) and a forward primer and reverse primer located in exons 3 and 5 of the human RHO gene, respectively. For the experiments in WERI-Rb-1 cells and zebrafish larvae, the target region was amplified from the synthesized human or zebrafish cDNA using Q5 High-Fidelity DNA Polymerase (#M0491L, New England Biolabs, Ipswich, MA, USA). For the exon skipping experiments in HEK293T cells and WERI-Rb-1 cells, primers amplifying GAPDH using Taq polymerase (M0491L, New England Biolabs, Ipswich, MA) were employed as a control. All primer sequences are listed in Table S2 . Amplified fragments were separated on a 1% agarose gel and sequence verified by Sanger sequencing.
4
Error-Prone PCR for CamR Randomization
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Random mutagenesis libraries were created by amplifying the coding region of the CamR gene with error-prone polymerase chain reaction. To perform this mutagenesis protocol, 50 ng of the template gene was mixed with 2 uL of 2 uM forward and reverse primers, 3 uL of Taq polymerase (New England BioLabs, Ipswich, MA, USA), 1 uM of 50 mM MnCl 2 , 41 uL of water, and 50 uL of a custom buffer (300 uL 10x Taq polymerase buffer, 16.5 uL 1 M MgCl 2 , 15 uL 10 mg/mL bovine serum albumin, 6 uL 100 mM dGTP, 11 uL 100 mM dATP, 12 uL 100 mM dCTP, 40.5 uL 100 mM dTTP, and 1099 uL water). The thermal cycling procedure is as follows: 94 °C for 30 seconds, 55C for 30 seconds, 72C for one minute, repeated for 25 cycles. Libraries were cloned into the pCamR plasmid using Gibson assembly and E. coli DH10B bearing pSELIScamr was transformed with the resulting library. Transformation efficiency always exceeded 10 6 for each round of selection, indicating several fold coverage of the library. Transformed cells were grown in LB media overnight at 37°C in carbenicillin and chloramphenicol.
5
PCR Detection of Bonamia exitiosa Actin
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Degenerate primers for actin amplification in Rhizopods (Longet et al. 2004 ) were tested in Whole-Genome amplified DNA of B. exitiosa purified cells (Table 2). The reaction was carried out in a volume of 50 µl with 2 mM of each dNTP, 2.5 units of Taq polymerase (New England Biolabs) using 300 ng of amplified DNA as template. Thermal cycling was 94ºC for 5 min, 40 cycles of 94ºC for 1 min of denaturing, 55ºC for 1 min of annealing and 72ºC for 2 min of extension, followed by 10 min of final extension at 72ºC. Bonamia ostrae DNA from infected oysters and distilled water were used as positive and negative controls, respectively. Amplification of actin gene from gills of Bonamia sp. infected oysters.
Specific primers (BeActI-F/BeActI-R, Table 2) for B. exitiosa actin amplification were designed in a region with low similarity to B. ostreae actin sequences. These primers amplified a product of 220 pairs of bases and were used to detect B. exitosa actin on genomic DNA from gills of Bonamia sp. infected oysters (Table 1). PCR reactions were performed in a volume of 25 µl containing 2 mM nucleotides, 1.5 units of Taq polymerase (New England Biolabs) and 100 ng of genomic material. Thermal cycling was 94ºC for 5 min, 30 cycles of 94ºC for 1 min of denaturing, 60ºC for 1 min of annealing and 72ºC for 2 min of extension, followed by 10 min of final extension at 72ºC.
Specific primers (BeActI-F/BeActI-R, Table 2) for B. exitiosa actin amplification were designed in a region with low similarity to B. ostreae actin sequences. These primers amplified a product of 220 pairs of bases and were used to detect B. exitosa actin on genomic DNA from gills of Bonamia sp. infected oysters (Table 1). PCR reactions were performed in a volume of 25 µl containing 2 mM nucleotides, 1.5 units of Taq polymerase (New England Biolabs) and 100 ng of genomic material. Thermal cycling was 94ºC for 5 min, 30 cycles of 94ºC for 1 min of denaturing, 60ºC for 1 min of annealing and 72ºC for 2 min of extension, followed by 10 min of final extension at 72ºC.
6
Nucleic Acid Extraction and Purification
7
Aptamer Selection and Protein Binding Assays
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Dithiothreitol (DTT), NaCl, MgCl2, 1× PBS buffer, glacial acetic acid, hydrochloric acid, DMSO, titanium (IV) isopropoxide, guanidine HCl (Gu-HCl) and potassium chloride (KCl) were obtained from Fisher Scientific. Polyvinylpyrrolidone (PVP) was acquired from Sigma-Aldrich. Tris base, EDTA and ethanol (EtOH) was obtained from Acros Organics, Promega and Decon Labs, respectively. The 5× Taq polymerase and the 25-mM magnesium chloride (MgCl2) solution used for PCR were from New England Biolabs. The ssDNAs, including the random libraries used for SELEX, the anti-IgE aptamer, as well as those found binding to DNMT1 (Supplementary Tables S1 and S3 ) were attained from Integrated DNA Technologies, Inc. Beta-casein, cytochrome C, ovalbumin, and hemoglobin were purchased from Sigma-Aldrich. IgE was acquired from Athens Research & Technology, Inc.
8
Coral Microbiome 16S rRNA Profiling
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DNA was extracted from coral tissues using the UltraClean Soil DNA Kit (Mo Bio; Carlsbad, CA, United States) following the manufacturer’s instructions for maximum yield. The extracted genomic DNA was used for PCR amplifications of V3–V4 region of the 16S rRNA gene by using the following universal primers: Pro341F (CCTACGGGNBGCASCAG) (Takahashi et al., 2014 (link)) and Bact805R (GACTACHVGGGTATCTAATCC) (Herlemann et al., 2011 (link)). Each PCR mixture contained 5 μl of 10x PCR reaction buffer (Invitrogen), 1.5 μl of 50 mM MgCl2, 1 μl 10 mM dNTP mixture, 1 μl of 100 μM of each primer, 1 units of Taq polymerase, 3 μl of BSA (New England BioLabs), sterile MilliQ water up to 50 μl and 10 ng of DNA. Negative controls (with no template DNA) were included to assess potential contamination of reagents. The amplification products were purified with the GeneJET PCR purification kit (Fermentas, EU), quantified using the Qubit Kit (Invitrogen), and the quality (integrity and presence of a unique band) was confirmed by 1% agarose gel electrophoresis.
9
Competitive PCR for CPEB2 Isoform Analysis
10
Confirming Transgene Placement and Homoplasmy in Transplastomic Strains
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To confirm the accurate transgene placement and to assess the homoplasmy of the engineered transplastomic strains, PCR reactions were performed using sets of primers listed in Additional file 1 : Table S1. Wild-type and transplastomic cells were treated with 10 mM Tris–EDTA lysis buffer and boiled for 10 min in preparation for PCR tests [28 (link)]. Taq polymerase (NEB, Ipswich, MA) was used in all PCR reactions for 35 cycles following the manufacturer’s instructions. Gene-specific primers were used to confirm the proper transgene insertion. To confirm the homoplasmy of the transformants, Fwd1 and Rev1 primers (Additional file 1 : Table S1) were designed to amplify a sequence at the insertion site that is disrupted if the homologous recombination is successful.
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