In order to generate the libraries of mutagenized tet(A), tet(K), tet(M), and tet(X) sequences, 10 separate error-prone PCRs per gene were run with AmpliTaq Gold DNA polymerase (Applied Biosystems) in mutagenic buffer (29 (link)). In short, a 50-μl PCR mixture contained approximately 10 ng of linearized plasmid DNA, 1× AmpliTaq Gold PCR buffer (Applied Biosystems), 1.5 mM MgCl2 (Applied Biosystems), 0.2 mM deoxynucleoside triphosphates (dNTPs; Thermo Scientific), 0.3 μM forward primer and 0.3 µM reverse primer (see Table S1 in the supplemental material), and 5 U of DNA polymerase. In addition, the PCR mixture was supplemented with 4 or 6 μl of mutagenic buffer (4 mM dCTP [Thermo Scientific], 4 mM dTTP [Thermo Scientific], 27.5 mM MgCl2 [Thermo Scientific], and 2.5 mM MnCl2) to vary the mutation rate. The PCR mixture was subjected to 2 min of initial denaturation at 94°C followed by 25 cycles of 1 min of denaturation at 94°C and 2 min of annealing and elongation at 72°C. A final elongation step was performed for 10 min at 72°C. After confirmation of PCR products with agarose gel electrophoresis, 10 reaction products were pooled to form the tet sequence library. Two sequence libraries (using 4 or 6 μl of mutagenic buffer) were generated per Tet determinant.
Mgcl2
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Lithuania, Canada, Netherlands, India, France, Portugal
Magnesium chloride (MgCl2) is an inorganic compound that is commonly used in laboratory settings. It is a white, crystalline solid that is soluble in water and other polar solvents. Magnesium chloride is a versatile compound that can be used in various applications, including as a desiccant, a coagulant, and a source of magnesium ions.
Automatically generated - may contain errors
Lab products found in correlation
Dntps, by Thermo Fisher Scientific (64 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (54 mentions)
Cacl2, by Thermo Fisher Scientific (48 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (46 mentions)
Pcr buffer, by Thermo Fisher Scientific (39 mentions)
Potassium chloride (kcl), by Thermo Fisher Scientific (33 mentions)
Taq polymerase, by Thermo Fisher Scientific (30 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (24 mentions)
Hepes, by Thermo Fisher Scientific (21 mentions)
Dntp mix, by Thermo Fisher Scientific (18 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (16 mentions)
Random hexamer, by Thermo Fisher Scientific (14 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (14 mentions)
Glycerol, by Thermo Fisher Scientific (14 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (11 mentions)
Tris hcl, by Thermo Fisher Scientific (11 mentions)
Bovine serum albumin (bsa), by Merck Group (11 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (10 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (10 mentions)
467 protocols using mgcl2
1
Error-Prone PCR for Tet Determinant Libraries
2
Nested PCR Amplification of DNA and cDNA
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Extracted DNA and cDNA samples were amplified by nested PCR in triplicate. Briefly, touch-up gradient PCR using primers SISP3 and SIS3 (Table S1) was used as the first-round amplification in a 50 μL reaction mix containing 6.5 μL template, 1 × Taq buffer (Applied Biosystems), 2 mM MgCl2 (Applied Biosystems), 0.5 mM dNTP (Sigma-Aldrich), 1 U Taq DNA polymerase (Roche Diagnostics), and 0.8 μM of each primer. The PCR reaction was performed for one cycle at 94 °C for 3 min, followed by 12 cycles touch-up PCR with 94 °C for 30 s and 20 °C for 190 s (2 °C increase per cycle), followed by 30 cycles of 94 °C for 30 s, 48 °C for 30 s, and 68 °C for 2 min, and with a 5 min final extension at 68 °C. Five microliters of the first-round PCR product were further amplified with the primers SISP2 and SIS2 (Table S1) in a 50 μL reaction mix containing 1 × Taq buffer (Applied Biosystems), 2 mM MgCl2 (Applied Biosystems), 0.5 mM dNTP (Sigma-Aldrich), 1 U Taq DNA polymerase (Roche Diagnostics), and 0.8 μM of each primer. The PCR was performed with an initial denaturation at 94 °C for 3 min followed by 45 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 2 min, and with a 5 min final extension at 68 °C. The triplicates were pooled, and the PCR products were visualized by 1.5% agarose gel electrophoresis.
3
Quantitative Real-Time RT-PCR Assay
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cDNAs were diluted 1:20 in H2O. Each 10 µL RT-PCR contained 2.5 µL of diluted cDNA, 1 µL 10 X Choice Taq buffer (Denville Scientific, Cat# C775Y44), 1X SYBR Green I (Invitrogen), 0.6 µL MgCl2 (50mM MgCl2, Invitrogen), 1 µL dNTP mix (2mM each dNTP, OMEGA BIO-TEK, Cat#101414-958), 1U Choice Taq (Denville Scientific, Cat# C775Y44), 0.8 µL primers (mix with 5µM each primer). Real-time RT-PCR analysis was carried out using a LightCycler® 480 Instrument II (Roche Diagnostics, USA) with the following cycling conditions: 95C 3”, 57C 20’, 72C 20’ for 50 cycles. Melt curves and gel electrophoresis were used to confirm the presence of a single amplification product of the correct size in each reaction. For all primer sets, a standard dilution curve was prepared using cDNAs pooled from all samples (SI Appendix, Table S7 ). Relative cDNA levels were calculated using the best-fit curve from the standard dilution of each primer set using supplied software from Roche and then normalized against the internal reference gene cDNA signal. Each reaction was run with three biological and two technical replicates.
4
Reverse Transcription Protocol for mRNA
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For the reverse transcription, a reaction mix was prepared with a final concentration of 2.5 µM random hexamers (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania), 1 mM of deoxyribonucleotide triphosphate (dNTPs) nucleotides (Eurogentec, Cologne, Germany), and 5 mM of MgCl2 (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania) in 1 x PCR buffer (50 mM KCl, 20 mM Tris-HCl, 5 mM MgCl2 pH 8.4; Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania). This was completed by 50 I.U. murine leukemia virus (MuLV) reverse transcriptase (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania) and 20 I.U. RNase-Inhibitor (Applied Biosystems by Thermo Fisher Scientific, Schwerte, Germany). Then, 11 µL of isolated mRNA was added to the mastermix solution. Finally, Ampuwa® water (KabiPac, Fresenius Kabi, Bad Homburg, Germany) was added to reach 20 µL total reaction volume.
Reverse transcription was performed for 10 min at 25 °C and for 60 min at 42 °C, followed by a denaturation step for 5 min at 99 °C. Finally, the samples were kept on ice and were immediately used for quantitative PCR (q-PCR) analysis.
Reverse transcription was performed for 10 min at 25 °C and for 60 min at 42 °C, followed by a denaturation step for 5 min at 99 °C. Finally, the samples were kept on ice and were immediately used for quantitative PCR (q-PCR) analysis.
5
Single-Nucleus ATAC-Seq Library Preparation
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Cells were collected in 1.5 mL centrifuge tube and added lysis buffer which consisted of 10 mM Tris-HCl pH7.5 (Thermo Fisher Scientific, USA), 10 mM NaCl (Thermo Fisher Scientific, USA), 3 mM MgCl2 (Thermo Fisher Scientific, USA), 0.1% Tween-20 (Sigma, USA), 0.1% NP-40 (Roche, Switzerland), 0.01% Digitonin (Sigma, USA), and 1% bovine serum albumin (BSA, BBI, UK). Then the lysates were centrifuged at 500 g for 5 min at 4°C and the supernatant was discarded. The obtained nuclei were then washed three times with ATAC wash buffer (10 mM Tris-HCl pH7.5 (Thermo Fisher Scientific, USA), 10 mM NaCl (Thermo Fisher Scientific, USA), 3 mM MgCl2 (Thermo Fisher Scientific, USA), 0.1% Tween-20 (Sigma, USA), 1% bovine serum albumin (BSA, BBI, UK)). Nuclei were then transposed using transposase (BGI, China) and were resuspended in nuclear resuspension buffer. Transposed single-nucleus suspensions were converted to barcoded scATAC-seq libraries, through procedures including droplet encapsulation, pre-amplification, emulsion breakage, capture beads collection, DNA amplification and purification. The libraries were sequenced on the ultra-high-throughput DIPSEQ T1 sequencer with PE 50-bp read length.
6
Single-cell RNA Extraction and Amplification
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Ten cells were collected in individual PCR tubes containing 5 µL PBS 1× following cell sorting on a FACSAria III U equipped with an automatic cell deposition unit (ACDU, BD Biosciences, San Diego, USA). Cells were lysed by freezing at −80 °C, followed by heating to 65 °C for 2 min.
After cooling at 4 °C, RNA was specifically reverse transcribed by adding 10 μL of a mix containing 0.13 μM RT primer, 1× PCR Buffer (Thermo), 3.3 mM MgCl2 (Thermo), 1 mM dNTPs (GE Healthcare), 40 units of RNaseOUT (Thermo), and 35 units of MuLV Reverse Transcriptase (Thermo), in a 15 μL reaction volume for 1 h at 37 °C and then incubated for 3 min at 95 °C for inactivation of reverse transcriptase. cDNA generated were dilute with 30 µL of 1× PCR Buffer.
A 4 µL aliquot of diluted cDNA was then amplified: first, a denaturation step at 95 °C for 10 min and then 54 amplification cycles (30 s at 95 °C, 45 s at 70 °C, and 60 s at 72 °C, with the hybridization temperature decreased from 70 to 60 °C during the six first cycles). The PCR mix contained 0.25 μM of each primer (Reverse and Forward), 1× PCR Buffer (Thermo), 2 mM MgCl2 (Thermo), 0.25 mM dNTPs (Thermo), and 0.5 units of AmpliTaq Gold DNA Polymerase (Thermo), in 20 μL reaction volume. PCR products were detected on a 1.5% agarose ethidium bromide gel. Primer sequences are described inTable S7 .
After cooling at 4 °C, RNA was specifically reverse transcribed by adding 10 μL of a mix containing 0.13 μM RT primer, 1× PCR Buffer (Thermo), 3.3 mM MgCl2 (Thermo), 1 mM dNTPs (GE Healthcare), 40 units of RNaseOUT (Thermo), and 35 units of MuLV Reverse Transcriptase (Thermo), in a 15 μL reaction volume for 1 h at 37 °C and then incubated for 3 min at 95 °C for inactivation of reverse transcriptase. cDNA generated were dilute with 30 µL of 1× PCR Buffer.
A 4 µL aliquot of diluted cDNA was then amplified: first, a denaturation step at 95 °C for 10 min and then 54 amplification cycles (30 s at 95 °C, 45 s at 70 °C, and 60 s at 72 °C, with the hybridization temperature decreased from 70 to 60 °C during the six first cycles). The PCR mix contained 0.25 μM of each primer (Reverse and Forward), 1× PCR Buffer (Thermo), 2 mM MgCl2 (Thermo), 0.25 mM dNTPs (Thermo), and 0.5 units of AmpliTaq Gold DNA Polymerase (Thermo), in 20 μL reaction volume. PCR products were detected on a 1.5% agarose ethidium bromide gel. Primer sequences are described in
7
Nuclease Treatment and Nucleic Acid Extraction
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Before nucleic acid extraction, 400 μL of the dissolved pellet was treated with 50 U Benzonase nuclease (Sigma-Aldrich, St. Louis, MO, USA) and 1.25 mM MgCl2 (Applied Biosystems, Foster City, CA, USA) and incubated at 37 °C for 1 h. Thereafter EDTA was added to a final concentration of 50 mM to inhibit the nuclease activity. DNA was extracted from 200 μL of each treated sample using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and RNA was extracted from the remaining 200 μL of each sample using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany).
8
Plant DNA Barcoding and Sequencing
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Samples were tested using CBOL Plant Working Group9 (link) recommended DNA regions rbcL, and matK as well as new primers designed in this study. The selected loci were amplified by polymerase chain reaction (PCR) on a PTC–100 thermocycler (Bio-Rad). DNA was amplified in 20 μL reaction mixtures containing 1 U AmpliTaq Gold Polymerase with GeneAmp 106 PCR buffer II (100 mm Tris–HCl pH 8.3, 500 mm KCl) and 2.5 mm MgCl2 (Applied Biosystems), 0.2 mm dNTPs, 0.1 mm of each primer (0.5 mm for matK), and 20 ng template DNA. Amplified products were sequenced for new markers in both directions with the primers used for amplification, following the protocols of the University of Guelph Genomics facility (www.uoguelph.ca/~genomics ). Products from each specimen were cleaned using Sephadex columns and run on an ABI 3730 sequencer (Applied Biosystems). Bidirectional sequence reads were obtained for all the PCR products. Sequences were assembled using Sequencher 4.5 (Gene Codes Corp), and aligned manually using Bioedit version 7.0.9.
9
cDNA Synthesis and RT-PCR Analysis
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cDNA was synthesized from 1 μg total or copy RNA using M-MLV reverse transcriptase and combined oligo-dT and pdN6 priming in 20 μl (Promega, Madison, WI). PCR was performed on 2.5 μl cDNA using Taq polymerase, MgCl2 and buffer from Applied Biosystems (Bleiswijk, Netherlands). For primer sequences see Supplementary Table 2 , for RT-PCR results per sample see Supplementary Table 3 .
10
qRT-PCR Analysis of Differentiation Markers
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mRNA was isolated from U937 cells (3×106 cells/differentiation time point) according to the user guide instructions of the RNeasy Kit (Qiagen, Venlo, Netherlands) and transcribed into cDNA (RNase Inhibitor, dNTP mix, MuLV Reverse Transcriptase, Oligo d(T)16, MgCl2 and Tag buffer: Applied Biosystems, Life Technologies, Carlsbad, CA, USA) in an Eppendorf Mastercycler personal (10 min 20°C, 20 min. 42°C, 5 min 95°C). Following primers were used for RT-PCR, using 1.5µl cDNA and 7.5µl SYBR@green (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) master mix and 0.375µl of each primer in a total volume of 15µl/sample:
human GAPDH
5′-GGCCTCCAAGGAGTAAGACC-3′ ,
3′-AGGGGTCTACATGGCAACTG-5′ ,
human CD14
5′-AGCCTAGACCTCAGCCACAA-3′ ,
3′-CTTGGCTGGCAGTCCTTTAG-5′ ,
human CD11b
5′-CCAGACGGAGACCAAAGTG-3′ ,
3′-GTCCTTGTATTGCCGCTTG-5′ , (VBC Biotech, Vienna, Austria) human PCI
5′- AGGCAAGATTGTGGACTTG-3′ ,
3′- GTTCCGGTCCAGGAGGTAGT-5′ (Invitrogen, Life Technologies, Carlsbad, CA, USA) and run on a Real Time PCR System (Applied Biosystems StepOnePlus) using FAST PCR 96-well plate, sealed with optically clear sealing tape (Sarstedt, Nümbrecht, Germany) with the fast program (40 cycles starting 95°C 20 s, 95°C 3 s, 60°C 30 s, and a melting curve of 95°C 15 s, 60°C 60 s, 95°C 15 s).
human GAPDH
human CD14
human CD11b
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