psti hf  (New England Biolabs)


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    Name:
    PstI HF
    Description:
    PstI HF 50 000 units
    Catalog Number:
    r3140l
    Price:
    269
    Size:
    50 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs psti hf
    PstI HF
    PstI HF 50 000 units
    https://www.bioz.com/result/psti hf/product/New England Biolabs
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    psti hf - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development"

    Article Title: Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

    Journal: bioRxiv

    doi: 10.1101/2020.06.22.165704

    Maps of doxycycline (dox)-inducible plasmids and over-expression analyses. A) Map of the pPID2 piggyBac cloning vector showing insulators in magenta; a DNA nuclear targeting sequence (DTS) in orange; multicloning sites (MCS) in purple; bacterial origin of replication (Ori) in cyan; bacterial β-lactamase (Bla) resistance gene (AmpR) in red; and piggyBac ITRs, IRES, and P2A sequences in grey. B) Map of the pPIDNB piggyBac dox-inducible vector showing restriction sites for cloning, coding sequences in yellow, terminator sequences in brown, and promoter sequences in green. pPIDNB constitutively expresses mNeonGreen (GFP) and coding sequences can be cloned into the plasmid under a bidirectional tetracycline (tet) inducible promoter using the AflII and PstI restriction sites. mScarlet-I, a red fluorescent protein (RFP), is expressed on the alternate side of the bidirectional tet promoter. C) Map of the pPIDNB2 vector, which is identical to pPIDNB except that GFP is localized to the nucleus using histone H2B. D) DF-1 cells transfected with pPIDNB constitutively express GFP and differentially express RFP in response to varying concentrations of dox after 24 hours. E) RFP-positive (i.e., dox-induced) cells relative to total number of GFP-positive (i.e., transfected) cells. F) dox induction was measured in DF-1 cells on the mRNA and G) protein levels for Cxcl14 (n = 2 for each group except n = 1 for the 2.5 ng/ml treatment; and mRNA level for H) Gas1 (n = 4 for each group). Levels are relative to 0 ng/ml of dox and normalized to 18S for mRNA and β-Actin for protein. I) Over-expression of Runx2 and J) Mmp13 with pPIDNB. DF-1 cells were transfected with control (cntrl) empty pPIDNB or pPIDNB plus Runx2 or Mmp13 coding sequence and treated with 50 ng/ml of dox for 24 hours. mRNA levels were normalized using 18S and protein using β-Actin. Representative WBs are shown below. N = 4 for each group. Error bars represent SEM. (*p
    Figure Legend Snippet: Maps of doxycycline (dox)-inducible plasmids and over-expression analyses. A) Map of the pPID2 piggyBac cloning vector showing insulators in magenta; a DNA nuclear targeting sequence (DTS) in orange; multicloning sites (MCS) in purple; bacterial origin of replication (Ori) in cyan; bacterial β-lactamase (Bla) resistance gene (AmpR) in red; and piggyBac ITRs, IRES, and P2A sequences in grey. B) Map of the pPIDNB piggyBac dox-inducible vector showing restriction sites for cloning, coding sequences in yellow, terminator sequences in brown, and promoter sequences in green. pPIDNB constitutively expresses mNeonGreen (GFP) and coding sequences can be cloned into the plasmid under a bidirectional tetracycline (tet) inducible promoter using the AflII and PstI restriction sites. mScarlet-I, a red fluorescent protein (RFP), is expressed on the alternate side of the bidirectional tet promoter. C) Map of the pPIDNB2 vector, which is identical to pPIDNB except that GFP is localized to the nucleus using histone H2B. D) DF-1 cells transfected with pPIDNB constitutively express GFP and differentially express RFP in response to varying concentrations of dox after 24 hours. E) RFP-positive (i.e., dox-induced) cells relative to total number of GFP-positive (i.e., transfected) cells. F) dox induction was measured in DF-1 cells on the mRNA and G) protein levels for Cxcl14 (n = 2 for each group except n = 1 for the 2.5 ng/ml treatment; and mRNA level for H) Gas1 (n = 4 for each group). Levels are relative to 0 ng/ml of dox and normalized to 18S for mRNA and β-Actin for protein. I) Over-expression of Runx2 and J) Mmp13 with pPIDNB. DF-1 cells were transfected with control (cntrl) empty pPIDNB or pPIDNB plus Runx2 or Mmp13 coding sequence and treated with 50 ng/ml of dox for 24 hours. mRNA levels were normalized using 18S and protein using β-Actin. Representative WBs are shown below. N = 4 for each group. Error bars represent SEM. (*p

    Techniques Used: Over Expression, Clone Assay, Plasmid Preparation, Sequencing, Transfection

    2) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    3) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    4) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging

    Journal: Journal of Virology

    doi: 10.1128/JVI.00840-18

    ORF68 PTC virus displays a genome cleavage defect. (A) Diagram depicting the DNA cleavage assay protocol and the expected TR DNA laddering phenotype (or lack thereof) upon infection with WT or ORF68 PTC virus. (B) Southern blot of the PstI-HF digested DNA with a TR probe from cells infected with WT, ORF68 PTC , or ORF68 PTC -MR virus.
    Figure Legend Snippet: ORF68 PTC virus displays a genome cleavage defect. (A) Diagram depicting the DNA cleavage assay protocol and the expected TR DNA laddering phenotype (or lack thereof) upon infection with WT or ORF68 PTC virus. (B) Southern blot of the PstI-HF digested DNA with a TR probe from cells infected with WT, ORF68 PTC , or ORF68 PTC -MR virus.

    Techniques Used: DNA Cleavage Assay, DNA Laddering, Infection, Southern Blot

    5) Product Images from "The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement"

    Article Title: The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004680

    Chromosomal regions where Smc5/6 accumulates after Top2 inhibition show no sign of persistent replication or recombination intermediates. ( A ) Chromosomal localization of Smc6-FLAG as determined by ChIP-seq at two loci, UBP10-MRPL19 and MPP10-YJR003C , showing abundant Smc6-FLAG binding in top2-4 . The lowest panel shows a ChIP-seq map from a control experiment performed on cells lacking FLAG-tagged proteins. Panel details and cellular growth conditions are as described in the legend of Figure 2 . In the top panel describing genomic features, arrows and chromosomal positions denote the restriction sites for PstI , used to produce the analyzed fragments. ( B ) Two-dimensional gel electrophoresis of UBP10-MRPL19 ( left ) and MPP10-YJR003C ( right ) in wild-type and top2-4 cells. Cell cycle progression monitored by fluorescence-activated cell sorting (FACS) is shown below and time-points of sample preparation are indicated. Membranes were first probed against UBP10-MRPL19 ( left ), then stripped and re-probed against MPP10-YJR003C ( right ), leaving some residual signal from UBP10-MRPL19 in the MPP10-YJR003C blots (white arrows). ( C ) Two-dimensional gel electrophoresis after DNA isolation using CTAB-extraction to preserve X-shaped molecules, of ARS305 ( left ) and UBP10-MRPL19 ( right ) in wild-type and top2-4 cells. The ARS305 containing fragment was produced by digestion using EcoRI and HindIII , and is a positive control for X-shaped molecule isolation (white arrowheads). Cell cycle progression monitored by FACS is shown below and indicates the time-points of sample preparation.
    Figure Legend Snippet: Chromosomal regions where Smc5/6 accumulates after Top2 inhibition show no sign of persistent replication or recombination intermediates. ( A ) Chromosomal localization of Smc6-FLAG as determined by ChIP-seq at two loci, UBP10-MRPL19 and MPP10-YJR003C , showing abundant Smc6-FLAG binding in top2-4 . The lowest panel shows a ChIP-seq map from a control experiment performed on cells lacking FLAG-tagged proteins. Panel details and cellular growth conditions are as described in the legend of Figure 2 . In the top panel describing genomic features, arrows and chromosomal positions denote the restriction sites for PstI , used to produce the analyzed fragments. ( B ) Two-dimensional gel electrophoresis of UBP10-MRPL19 ( left ) and MPP10-YJR003C ( right ) in wild-type and top2-4 cells. Cell cycle progression monitored by fluorescence-activated cell sorting (FACS) is shown below and time-points of sample preparation are indicated. Membranes were first probed against UBP10-MRPL19 ( left ), then stripped and re-probed against MPP10-YJR003C ( right ), leaving some residual signal from UBP10-MRPL19 in the MPP10-YJR003C blots (white arrows). ( C ) Two-dimensional gel electrophoresis after DNA isolation using CTAB-extraction to preserve X-shaped molecules, of ARS305 ( left ) and UBP10-MRPL19 ( right ) in wild-type and top2-4 cells. The ARS305 containing fragment was produced by digestion using EcoRI and HindIII , and is a positive control for X-shaped molecule isolation (white arrowheads). Cell cycle progression monitored by FACS is shown below and indicates the time-points of sample preparation.

    Techniques Used: Inhibition, Chromatin Immunoprecipitation, Binding Assay, Two-Dimensional Gel Electrophoresis, Electrophoresis, Fluorescence, FACS, Sample Prep, DNA Extraction, Produced, Positive Control, Isolation

    6) Product Images from "Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus"

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.105924

    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.
    Figure Legend Snippet: ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Techniques Used: Polymerase Chain Reaction, Produced, Southern Blot, Sequencing, Plasmid Preparation

    Related Articles

    Clone Assay:

    Article Title: Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development
    Article Snippet: .. Following confirmation of cloning of full length coding sequences by Sanger sequencing, Runx2 , Mmp13 , Cxcl14 , and Gas1 were cloned into pEPIC1.1 digested with AflII (NEB, R0520S) and EcoRI (NEB, R3101S) or pPIDNB digested with AflII (NEB, R0520S) and PstI (NEB, R3140S) using NEBuilder HiFi DNA Assembly Master Mix. .. All constructs were verified by Sanger sequencing and midipreped for electroporation and transfection using PureLink Fast Low-Endotoxin Midi Kit (Invitrogen, A36227).

    Southern Blot:

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus
    Article Snippet: .. Southern blotting 5 µg undigested CRF-CA D citri DNA or 5 µg CRF-CA D. citri DNA digested overnight with PstI-HF and HindIII-HF (New England Biolabs, Ibswich, Massachusetts) was electrophoresed in a 0.8% agarose/0.5x TAE gel and the gel was prepared for transfer by incubation in 0.25 M HCl for 30 minutes, then 0.5 M NaCl, 0.5 M NaOH for 30 minutes, and then 1.5 M NaCl, 0.5 M Tris-HCl pH 7 for 30 minutes. .. DNA was then transferred to an Amersham Hybond Nx membrane (GE Life Sciences, Boston, Massachusetts) for 24 hours in 20X SSC using the capillary method.

    Purification:

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Electrophoresis:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging
    Article Snippet: .. DNA (10 μg) was digested with PstI-HF (New England BioLabs) overnight then separated by electrophoresis in a 0.7% agarose 1× TBE gel stained with SYBRsafe. .. The DNA was transferred to a NYTRAN-N membrane (GE Healthcare) by capillary action in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) overnight and cross-linked to the membrane in a StrataLinker 2400 (Stratagene) using the AutoUV setting.

    Polymerase Chain Reaction:

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Incubation:

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus
    Article Snippet: .. Southern blotting 5 µg undigested CRF-CA D citri DNA or 5 µg CRF-CA D. citri DNA digested overnight with PstI-HF and HindIII-HF (New England Biolabs, Ibswich, Massachusetts) was electrophoresed in a 0.8% agarose/0.5x TAE gel and the gel was prepared for transfer by incubation in 0.25 M HCl for 30 minutes, then 0.5 M NaCl, 0.5 M NaOH for 30 minutes, and then 1.5 M NaCl, 0.5 M Tris-HCl pH 7 for 30 minutes. .. DNA was then transferred to an Amersham Hybond Nx membrane (GE Life Sciences, Boston, Massachusetts) for 24 hours in 20X SSC using the capillary method.

    Sequencing:

    Article Title: Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development
    Article Snippet: .. Following confirmation of cloning of full length coding sequences by Sanger sequencing, Runx2 , Mmp13 , Cxcl14 , and Gas1 were cloned into pEPIC1.1 digested with AflII (NEB, R0520S) and EcoRI (NEB, R3101S) or pPIDNB digested with AflII (NEB, R0520S) and PstI (NEB, R3140S) using NEBuilder HiFi DNA Assembly Master Mix. .. All constructs were verified by Sanger sequencing and midipreped for electroporation and transfection using PureLink Fast Low-Endotoxin Midi Kit (Invitrogen, A36227).

    Staining:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging
    Article Snippet: .. DNA (10 μg) was digested with PstI-HF (New England BioLabs) overnight then separated by electrophoresis in a 0.7% agarose 1× TBE gel stained with SYBRsafe. .. The DNA was transferred to a NYTRAN-N membrane (GE Healthcare) by capillary action in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) overnight and cross-linked to the membrane in a StrataLinker 2400 (Stratagene) using the AutoUV setting.

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  • 97
    New England Biolabs hf psti high fidelity
    Adapter Design, PCR amplification of fragments. 1) The ligation product of a genomic DNA fragment (black) containing a <t>PstI</t> restriction site and a <t>MspI</t> restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with “X”. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment.
    Hf Psti High Fidelity, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hf psti high fidelity/product/New England Biolabs
    Average 97 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    hf psti high fidelity - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    Image Search Results


    Adapter Design, PCR amplification of fragments. 1) The ligation product of a genomic DNA fragment (black) containing a PstI restriction site and a MspI restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with “X”. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment.

    Journal: PLoS ONE

    Article Title: Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach

    doi: 10.1371/journal.pone.0032253

    Figure Lengend Snippet: Adapter Design, PCR amplification of fragments. 1) The ligation product of a genomic DNA fragment (black) containing a PstI restriction site and a MspI restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with “X”. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment.

    Article Snippet: Restriction Digest: Genomic DNA (200 ng) was digested in 20 ul reaction volume of NEB Buffer 4 with 8 U of HF-PstI (High-Fidelity) and 8 U of MspI (New England BioLabs Inc., Ipswich, MA 01938).

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Generated, Synthesized

    Maps of doxycycline (dox)-inducible plasmids and over-expression analyses. A) Map of the pPID2 piggyBac cloning vector showing insulators in magenta; a DNA nuclear targeting sequence (DTS) in orange; multicloning sites (MCS) in purple; bacterial origin of replication (Ori) in cyan; bacterial β-lactamase (Bla) resistance gene (AmpR) in red; and piggyBac ITRs, IRES, and P2A sequences in grey. B) Map of the pPIDNB piggyBac dox-inducible vector showing restriction sites for cloning, coding sequences in yellow, terminator sequences in brown, and promoter sequences in green. pPIDNB constitutively expresses mNeonGreen (GFP) and coding sequences can be cloned into the plasmid under a bidirectional tetracycline (tet) inducible promoter using the AflII and PstI restriction sites. mScarlet-I, a red fluorescent protein (RFP), is expressed on the alternate side of the bidirectional tet promoter. C) Map of the pPIDNB2 vector, which is identical to pPIDNB except that GFP is localized to the nucleus using histone H2B. D) DF-1 cells transfected with pPIDNB constitutively express GFP and differentially express RFP in response to varying concentrations of dox after 24 hours. E) RFP-positive (i.e., dox-induced) cells relative to total number of GFP-positive (i.e., transfected) cells. F) dox induction was measured in DF-1 cells on the mRNA and G) protein levels for Cxcl14 (n = 2 for each group except n = 1 for the 2.5 ng/ml treatment; and mRNA level for H) Gas1 (n = 4 for each group). Levels are relative to 0 ng/ml of dox and normalized to 18S for mRNA and β-Actin for protein. I) Over-expression of Runx2 and J) Mmp13 with pPIDNB. DF-1 cells were transfected with control (cntrl) empty pPIDNB or pPIDNB plus Runx2 or Mmp13 coding sequence and treated with 50 ng/ml of dox for 24 hours. mRNA levels were normalized using 18S and protein using β-Actin. Representative WBs are shown below. N = 4 for each group. Error bars represent SEM. (*p

    Journal: bioRxiv

    Article Title: Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

    doi: 10.1101/2020.06.22.165704

    Figure Lengend Snippet: Maps of doxycycline (dox)-inducible plasmids and over-expression analyses. A) Map of the pPID2 piggyBac cloning vector showing insulators in magenta; a DNA nuclear targeting sequence (DTS) in orange; multicloning sites (MCS) in purple; bacterial origin of replication (Ori) in cyan; bacterial β-lactamase (Bla) resistance gene (AmpR) in red; and piggyBac ITRs, IRES, and P2A sequences in grey. B) Map of the pPIDNB piggyBac dox-inducible vector showing restriction sites for cloning, coding sequences in yellow, terminator sequences in brown, and promoter sequences in green. pPIDNB constitutively expresses mNeonGreen (GFP) and coding sequences can be cloned into the plasmid under a bidirectional tetracycline (tet) inducible promoter using the AflII and PstI restriction sites. mScarlet-I, a red fluorescent protein (RFP), is expressed on the alternate side of the bidirectional tet promoter. C) Map of the pPIDNB2 vector, which is identical to pPIDNB except that GFP is localized to the nucleus using histone H2B. D) DF-1 cells transfected with pPIDNB constitutively express GFP and differentially express RFP in response to varying concentrations of dox after 24 hours. E) RFP-positive (i.e., dox-induced) cells relative to total number of GFP-positive (i.e., transfected) cells. F) dox induction was measured in DF-1 cells on the mRNA and G) protein levels for Cxcl14 (n = 2 for each group except n = 1 for the 2.5 ng/ml treatment; and mRNA level for H) Gas1 (n = 4 for each group). Levels are relative to 0 ng/ml of dox and normalized to 18S for mRNA and β-Actin for protein. I) Over-expression of Runx2 and J) Mmp13 with pPIDNB. DF-1 cells were transfected with control (cntrl) empty pPIDNB or pPIDNB plus Runx2 or Mmp13 coding sequence and treated with 50 ng/ml of dox for 24 hours. mRNA levels were normalized using 18S and protein using β-Actin. Representative WBs are shown below. N = 4 for each group. Error bars represent SEM. (*p

    Article Snippet: Following confirmation of cloning of full length coding sequences by Sanger sequencing, Runx2 , Mmp13 , Cxcl14 , and Gas1 were cloned into pEPIC1.1 digested with AflII (NEB, R0520S) and EcoRI (NEB, R3101S) or pPIDNB digested with AflII (NEB, R0520S) and PstI (NEB, R3140S) using NEBuilder HiFi DNA Assembly Master Mix.

    Techniques: Over Expression, Clone Assay, Plasmid Preparation, Sequencing, Transfection

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation