The transcription start site for genes lvaR and lvaA were isolated using an adapted 5’ Race protocol from Schramm et al(Schramm et al., 2000 (link)). The RNA isolated from P. putida KT2440 was treated with the TURBO DNA-free™ Kit from Ambion® to remove any contaminating DNA. The Promega GoScript RT PCR kit was used to generate cDNA using 1 µL of a 10 µM gene specific oligo (JMR2 for lvaR and JMR287 for lvaA) instead of the random oligo mixture. Following the inactivation of the reverse transcriptase, the cDNA was purified using Qiagen PCR Purification kit. Tailing of the cDNA was achieved using the terminal deoxynucleotidyl transferase (TdT) enzyme from Thermo Scientific. The final reaction mixture contained 1× reaction buffer, 1 pmol cDNA fragments, 60 pmol dGTP or dCTP and 30 U TdT. The reaction was incubated at 37°C for 15 min and then quenched by heating to 70°C for 10 min and the tailed cDNA fragments cleaned up using a Qiagen PCR Purification kit. The tailed cDNA was amplified using GoTaq Green Master Mix with an annealing temperature of 55°C and an extension time of 30 sec. Primer GG318 was used for dGTP tailing and ALM244 was used for dCTP tailing. The reverse primer for lvaR was JMR150 and for lvaA was JMR296. The resulting PCR product was submitted for sequencing.
Pcr purification kit
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The PCR purification kit is a laboratory product designed to purify and concentrate DNA samples obtained from Polymerase Chain Reaction (PCR) amplification. The kit provides a streamlined process for removing unwanted components, such as primers, nucleotides, and salts, from the PCR reaction mixture, allowing for the recovery of high-quality, purified DNA samples.
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Lab products found in correlation
Gel extraction kit, by Qiagen (24 mentions)
Bioruptor, by Diagenode (21 mentions)
Bioruptor sonicator, by Diagenode (18 mentions)
T4 dna ligase, by New England Biolabs (17 mentions)
Pgem t easy vector, by Promega (15 mentions)
T4 dna ligase, by Thermo Fisher Scientific (15 mentions)
Miseq platform, by Illumina (14 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (13 mentions)
Qiaquick gel extraction kit, by Qiagen (13 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
Dneasy blood and tissue kit, by Qiagen (12 mentions)
Nanodrop, by Thermo Fisher Scientific (12 mentions)
Qiaprep spin miniprep kit, by Qiagen (10 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (10 mentions)
Nextseq 500, by Illumina (10 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Gel purification kit, by Qiagen (9 mentions)
Protein g dynabead, by Thermo Fisher Scientific (9 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (9 mentions)
1 229 protocols using pcr purification kit
1
Mapping Transcriptional Start Sites
2
Mapping Transcriptional Start Sites
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The transcription start site for genes lvaR and lvaA were isolated using an adapted 5’ Race protocol from Schramm et al(Schramm et al., 2000 (link)). The RNA isolated from P. putida KT2440 was treated with the TURBO DNA-free™ Kit from Ambion® to remove any contaminating DNA. The Promega GoScript RT PCR kit was used to generate cDNA using 1 µL of a 10 µM gene specific oligo (JMR2 for lvaR and JMR287 for lvaA) instead of the random oligo mixture. Following the inactivation of the reverse transcriptase, the cDNA was purified using Qiagen PCR Purification kit. Tailing of the cDNA was achieved using the terminal deoxynucleotidyl transferase (TdT) enzyme from Thermo Scientific. The final reaction mixture contained 1× reaction buffer, 1 pmol cDNA fragments, 60 pmol dGTP or dCTP and 30 U TdT. The reaction was incubated at 37°C for 15 min and then quenched by heating to 70°C for 10 min and the tailed cDNA fragments cleaned up using a Qiagen PCR Purification kit. The tailed cDNA was amplified using GoTaq Green Master Mix with an annealing temperature of 55°C and an extension time of 30 sec. Primer GG318 was used for dGTP tailing and ALM244 was used for dCTP tailing. The reverse primer for lvaR was JMR150 and for lvaA was JMR296. The resulting PCR product was submitted for sequencing.
3
Preparation of Dinucleosomes from 601 DNA
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The overall methodology of preparation of dinucleosomes was adapted with minor changes from a previously described protocol (McGinty et al., 2008 (link)). After PCR amplification of the 601 DNA constructs designed for nucleosome A and nucleosome B, DNA was purified using a Qiagen PCR purification kit and resuspended in water. Approximately 100 μg of 601 DNA was digested with 750 U of DraIII-HF (NEB, at 20,000 U/mL) in a total volume of 1 mL of 1 X CutSmart buffer (NEB) for 8 h at 37 °C. The 601 DNA was then purified again using a Qiagen PCR purification kit and resuspended in water to a concentration of 1 μg/μL.
4
Large-scale cDNA Amplification and Fractionation
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The large scale amplified cDNAs obtained with both synthesis protocols were pooled and column purified (Qiagen PCR Purification Kit, Qiagen, The Netherlands), run on a 1% agarose gel and three separate size ranges were fractionated: 1–2 kb, 2–3 kb, and over 3 kb. Each size fraction was extracted from the gel (Qiagen Gel Extraction Kit, Qiagen, The Netherlands), purified and amplified for additional eight PCR cycles with SMARTer or TeloPrime specific primer pairs, respectively. PCR products were again pooled and column purified (Qiagen PCR Purification Kit, Qiagen, Netherlands). Single Molecule Real Time (SMRT) bell libraries were prepared as recommended by Pacific Biosciences (Palo Alto, U.S.A). SMRT bell templates were bound to polymerase using the DNA polymerase binding kit P6 v2 primers.
5
Cas9 mRNA and gRNA Production
6
Glucose Starvation RNA Sequencing
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Total RNA extracted from control and 8 hr glucose starved cells was used as a template to create cDNA with superscript II (Invitrogen) according to the manufacturer’s instructions using gene-specific primers. The cDNA was purified using a PCR purification kit (Qiagen, Germany), followed by addition of polyG to the 5’-end using TdT enzyme (Promega) for 1 hr at 37°C. The reaction was terminated by heat inactivation for 15 min at 65°C, and the products were purified with the PCR purification kit (Qiagen). The modified cDNA was used as a template for PCR with Phusion polymerase (NEB) using nested reverse primers and forward PolyC primer. PCR products were run on 6% polyacrylamide gel.
7
Inverse PCR and Nested PCR Protocol
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Inverse PCR and subsequent nested PCRs were performed using GoTaq Flexi DNA polymerase (Promega). Genomic DNA was purified from isolated clones and 2 μg genomic DNA was digested with the restriction enzymes NheI, HindIII, PvuI, SgrAI, BsmI, BmtI, AgeI, EcoRI and MfeI overnight. The digested genomic DNA was purified by PCR purification kit (Qiagen). T4 DNA ligase (NEB) was used to self-ligate 250 ng of digested genomic DNA using 1 μL of enzyme in a 250 ul reaction volume to promote self-ligation with overnight incubation. Next day, ligated DNA was again purified using the PCR purification kit (Qiagen) and inverse PCR was performed with eluted DNA as follows: initial denaturation at 95°C for 2 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 8 min, and a final step of 72°C for 10 min using the primers EF_rev_474 and 67_mCherry_fwd listed in Supplementary Table S1 . Next, nested PCR was performed with 2 μL of inverse PCR product in a 50 μL reaction using same conditions with the EF_rev_104 and mCherry_fwd_597 primers listed in Supplementary Table S1 . PCR products were resolved on 0.8% agarose gels and amplified bands were sequenced.
8
5'/3' RACE Protocol for U2OS cells
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5’RACE (5’/3’ RACE kit 2nd generation Roche) was performed using total and ribosomal RNA from U2OS cells following manufacturers’ instructions with few modifications to the protocol. We added a denaturation step at 90°C for 2 minutes and an annealing step at 59°C for 30 minutes to the suggested first strand cDNA synthesis protocol using the primer 5.8S Rev (Table 1 ). Reverse transcription (RT) was carried out at 42°C for 1 hour. First strand cDNA was then purified using PCR purification kit (Qiagen). Poly(A) or poly(T) tailing of the first strand cDNA was then performed. Finally, a PCR using AccuPrime, a high fidelity Taq DNA polymerase (Invitrogen), was performed using 5.8S Rev primer (Table 1 ) and the degenerated oligo dT-anchor Primer of the kit in the case of poly(A) tailing or a degenerated oligo dA-anchor primer (IDT) in the case of poly(T) tailing. The PCR product was then purified using PCR purification kit (Qiagen) and Sanger sequenced.
9
Cloning of miR-888 Binding Sites
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An approximately 500-bp region surrounding each of the four miR-888 binding sites in the PR 3′UTR was amplified from ECC-1 genomic DNA using the Platinum PCR SuperMix, High Fidelity (Life Technologies; primer sequences in Table S5 ). PCR products were purified using the PCR Purification Kit (Qiagen, Valencia, CA) and subcloned into the pGEM-T Easy vector (Promega, Madison, WI). Insert sequences were subsequently cloned into the psiCHECK2 vector and transformed into TOP10 OneShot competent E. coli (Life Technologies).
An approximately 500-bp region surrounding each of the four miR-888 binding sites in the PR 3′UTR was amplified from ECC-1 genomic DNA using the Platinum PCR SuperMix, High Fidelity (Life Technologies; primer sequences in Table S5). PCR products were purified using the PCR Purification Kit (Qiagen, Valencia, CA) and subcloned into the pGEM-T Easy vector (Promega, Madison, WI). Insert sequences were subsequently cloned into the psiCHECK2 vector and transformed into TOP10 OneShot competent E. coli (Life Technologies).
An approximately 500-bp region surrounding each of the four miR-888 binding sites in the PR 3′UTR was amplified from ECC-1 genomic DNA using the Platinum PCR SuperMix, High Fidelity (Life Technologies; primer sequences in Table S5). PCR products were purified using the PCR Purification Kit (Qiagen, Valencia, CA) and subcloned into the pGEM-T Easy vector (Promega, Madison, WI). Insert sequences were subsequently cloned into the psiCHECK2 vector and transformed into TOP10 OneShot competent E. coli (Life Technologies).
10
Reverse Cross-Linking of Chromatin DNA
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10–30 μL of the sonicated chromatin samples were combined with 10 μL of 10 mg/mL Proteinase K (Sigma, P2308), 50 μL of 2x reverse cross-linking master mix (100 mM Tris pH 8.0, 600 mM NaCl, 1.0% SDS, 100 mM EDTA pH 8.0; kept at +37°C to avoid SDS precipitation) and brought to a final volume of 100 μL with water. Two reverse cross-linking procedures were used. In the standard procedure, samples were first incubated overnight at +60°C, combined with 10 μL of 1 mg/mL RNaseA (Qiagen, 1018048) the following morning and incubated for an additional 1 h at +60°C. The resulting DNA was purified with a PCR purification kit (Qiagen, 28104). In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104). The traditional overnight procedure was used in most cases, except for two samples that were prepared using the quick procedure, as indicated in text. The advantages of each procedure are discussed in the next section. Purified DNA was analysed on ethidium bromide stained agarose gels.
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