Plasmid Preparation:
Article Title: ALKBH5 regulates somatic cell reprogramming in a phase-specific manner
Article Snippet: .. The correct clone was used as entry clone and then the construct was shuttled to destination vector pLX301 (Addgene plasmid # 25895 ) For constitutive overexpression of either ALKBH5 or ALKBH5-HA, and pCW57.1 (Addgene plasmid # 41393 ) for dox-inducible overexpression using LR clonase (Thermo Fisher Scientific #11791020) based on the manufacturer's protocol. ..
Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Construct:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase-specific manner
Article Snippet: .. The correct clone was used as entry clone and then the construct was shuttled to destination vector pLX301 (Addgene plasmid # 25895 ) For constitutive overexpression of either ALKBH5 or ALKBH5-HA, and pCW57.1 (Addgene plasmid # 41393 ) for dox-inducible overexpression using LR clonase (Thermo Fisher Scientific #11791020) based on the manufacturer's protocol. ..
Over Expression:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase-specific manner
Article Snippet: .. The correct clone was used as entry clone and then the construct was shuttled to destination vector pLX301 (Addgene plasmid # 25895 ) For constitutive overexpression of either ALKBH5 or ALKBH5-HA, and pCW57.1 (Addgene plasmid # 41393 ) for dox-inducible overexpression using LR clonase (Thermo Fisher Scientific #11791020) based on the manufacturer's protocol. ..
Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Transduction:
Clone Assay:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Transformation Assay:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Polymerase Chain Reaction:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Sequencing:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Purification:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Virus:Article Title: ALKBH5 regulates somatic cell reprogramming in a phase specific manner
Article Snippet: Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to GatewayTM pDONRTM221 Vector (Invitrogen#12536017) using GatewayTM BP ClonaseTM II Enzyme mix (Invitrogen#11789020), then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. .. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation, the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus. .. Two short hairpins shRNA for targeting mAlkbh5 were annealed in annealing buffer by heating 10 minutes at 95C In PCR machine then cooling by gradual decreasing the temperature to 4C in 30 minutes, and then the annealed oligos were ligated using T4 DNA Ligase (5 U/μL) (Thermo Fisher Scientific #EL0011) to either pLKO.1 puro (Addgene Plasmid #8453) for constitutive knockdown or Tet-pLKO-puro (Addgene Plasmid #21915) for periodic knockdown which was pre-linearized with AgeI-HF (NEB # R3552L) and EcoRI-HF (NEB#R3101S) restriction enzymes, then transformed to One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Fisher #C737303), and several colonies were picked up and sent for sequencing.
Generated:Article Title: Comprehensive laboratory diagnosis of Fanconi anaemia: comparison of cellular and molecular analysis
Article Snippet: .. pLX301-FANCA, pLX301-FANCG and pLX301-FANCC were generated by Gateway cloning using the entry vectors pENTR223.1-FANCA (TransOMIC Technologies), pDONR221-FANCG (DNASU), and pDONR221-FANCC (DNASU), and the destination vector pLX301 (a kind gift from David Root, Addgene ID: 25895). .. Gateway cloning was carried out using the LR Clonase Enzyme mix (Thermo Scientific).
Cloning:Article Title: Comprehensive laboratory diagnosis of Fanconi anaemia: comparison of cellular and molecular analysis
Article Snippet: .. pLX301-FANCA, pLX301-FANCG and pLX301-FANCC were generated by Gateway cloning using the entry vectors pENTR223.1-FANCA (TransOMIC Technologies), pDONR221-FANCG (DNASU), and pDONR221-FANCC (DNASU), and the destination vector pLX301 (a kind gift from David Root, Addgene ID: 25895). .. Gateway cloning was carried out using the LR Clonase Enzyme mix (Thermo Scientific).
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