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.
Tdt enzyme
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TdT (Terminal deoxynucleotidyl Transferase) enzyme is a DNA polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl end of DNA strands in a template-independent manner. This enzyme is commonly used in various molecular biology and biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Tdt buffer, by Thermo Fisher Scientific (3 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Goscript rt pcr kit, by Promega (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Nbt bcip, by Roche (2 mentions)
Tunel assay, by Merck Group (1 mentions)
Exosap it express, by Thermo Fisher Scientific (1 mentions)
Ez 96 dna methylation direct magprep kit, by Zymo Research (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
5 race system for rapid amplification of cdna ends, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Biotinylated 16 dutp, by Roche (1 mentions)
8 protocols using tdt enzyme
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
TUNEL Assay for Apoptosis Detection
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TUNEL assay (CN: 17141/Millipore) was carried out according to the manufacturer’s instructions. For FFPE sections, samples were deparaffinized and rehydrated with ethanol wash. Next, sections were incubated with 20 µg/mL proteinase K (Thermo Fisher Scientific, Waltham, MA, USA, AM2548) for 20 min at 37 °C to remove formalin-generated crosslinks. For cultured neurons on coverslips, cells were fixed with methanol. Briefly, the samples were treated with TdT enzyme (Thermo Fisher Scientific, Waltham, MA, USA, EP0161), washed, and incubated with anti-digoxigenin conjugate to label apoptotic cells.
4
Whole-Genome Bisulfite Sequencing of Human Cells
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Human 3PN eight-cell embryos (three embryos were pooled as one biological replicate), 200 ng of purified gDNA of hES cells or h293T cells was subjected to bisulfite conversion and purification by using the EZ-96 DNA Methylation-Direct MagPrep kit (Zymo Research, cat. no. D5045) according to the manufacturer's protocol. Then, WGBS libraries were constructed via our recently published TAILS method 22, 47 . In brief, P5-N6-oligo1 (5′-CTACACGACGCTCTTCCGATCTN 6 -3′) was used for the first round of random priming in the presence of the Klenow exo(-) fragment (QIAGEN, cat. no. P7010-HC-L). The remaining oligonucleotides and dNTPs were removed by Exo-SAP IT Express (Applied Biosystems, cat. no. 75001) and the dC tailing step was performed with the TdT enzyme (Thermo Fisher, cat. no. EP0162). Then, a second round of priming was conducted by using P7-G6-oligo2 (5′-AGACGTGTGCTCTTCCGATCTG 6 HN-3′) in the presence of the Klenow exo(-) fragment. After one round of purification with AMPure XP beads, libraries were constructed by PCR amplification. Next, the libraries were purified and the size distribution was checked on the Fragment Analyzer. Finally, the WGBS libraries were sequenced on a NovaSeq 6000 sequencer with a 150-bp paired-end sequencing strategy.
5
5' RACE for cDNA Amplification
6
Detecting Apoptosis in Xenopus Embryos
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TUNEL assay of Xenopus embryos was performed according to the Harland protocol (Conlon lab) available in Xenbase. Stored embryos were rehydrated and washed in 1xPBS and incubated for 1 hour at RT in TdT buffer (Invitrogen). Then, 150 U/ml TdT enzyme (Invitrogen) and 0.1 µl of DdUTP (Roche) per 100 µl buffer were added to the buffer solution and the embryos were incubated overnight at RT. The next day, embryos were washed 2x1 hour at 65°C in 1 mM EDTA/PBS and in 1xPBS 4x1 hour at RT, followed by 2–10 min washes in 1xMAB. Then they were blocked in 2%BMB blocking solution for 1 hour at RT and incubated in a 1/3000 dilution of anti-digoxigenin AP antibody in BMB block for 4 hours RT or overnight at 4°C. Antibody was washed away by 5x1 hour washes in MAB. Endogenous phosphatases were blocked by 2x10 min washes in alkaline phosphatase buffer and then NBT/BCIP (Roche) was added to the embryos. Chromogenic reaction was stopped by a quick wash in 1XMAB and then the embryos were fixed overnight in 1xMEMFA at RT. The next day embryos were imaged after clearing in two parts Benzyl Benzoate and one part Benzyl Alcohol after dehydration (Murray's Clearing Medium) (2∶1 BB: BA).
7
Spatial Distribution of Apoptotic Cells After Ischemia
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To clarify the spatial distribution of apoptotic cells after cerebral ischemia, we performed terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining as previously described (n = 8 each) [1 (link)]. Fixed sections were incubated with NeuroPore (Trevigen) for 30 min. They were placed in 1 × terminal deoxynucleotidyl transferase (TdT) buffer (Invitrogen) with a TdT enzyme (Invitrogen) and biotinylated 16-dUTP (Roche Diagnostics) at 37 °C for 90 min. The avidin–biotin technique was applied and then the nuclei were counterstained with methyl green solution for 2 min. For more precise confirmation about SR-FLIVO-FMNP probe tracking within the apoptotic cells, high resolution transmission electron microscopy (HRTEM) was recorded for the cells located at ischemic and non-ischemic lesions.
8
TUNEL Assay for Xenopus Embryos
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TUNEL assay of Xenopus embryos was performed according to the Harland protocol (Conlon lab) available in Xenbase. Stored embryos were rehydrated and washed in 1xPBS and incubated for 1 hour at RT in TdT buffer (Invitrogen). Then, 150 U/ml TdT enzyme (Invitrogen) and 0.1 ml of DdUTP (Roche) per 100 ml buffer were added to the buffer solution and the embryos were incubated overnight at RT. The next day, embryos were washed 2x1 hour at 65uC in 1 mM EDTA/PBS and in 1xPBS 4x1 hour at RT, followed by 2-10 min washes in 1xMAB. Then they were blocked in 2%BMB blocking solution for 1 hour at RT and incubated in a 1/3000 dilution of anti-digoxigenin AP antibody in BMB block for 4 hours RT or overnight at 4uC. Antibody was washed away by 5x1 hour washes in MAB. Endogenous phosphatases were blocked by 2x10 min washes in alkaline phosphatase buffer and then NBT/BCIP (Roche) was added to the embryos. Chromogenic reaction was stopped by a quick wash in 1XMAB and then the embryos were fixed overnight in 1xMEMFA at RT. The next day embryos were imaged after clearing in two parts Benzyl Benzoate and one part Benzyl Alcohol after dehydration (Murray's Clearing Medium) (2:1 BB: BA).
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