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    New England Biolabs bsrgi
    Generation of AS knockout in P. berghei . ( a ) Double cross-over recombination strategy followed to generate Pb <t>ASKO.</t> ( b ) PCR analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: PCR with Pb AS-specific primers; Lane 2 and 4: PCR with Pb GAPDH-specific primers (control); Lane M: 1 kb DNA ladder. ( c ) RT-PCR analysis for RNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: RT-PCR with Pb AS-specific primers; Lane 2 and 4: RT-PCR with Pb GAPDH specific primers (control); Lane M: 1 kb DNA ladder. ( d ) Southern analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Transgenic plasmid (TP) was also included as control to rule out the presence of any episomes. Genomic DNA preparations and TP were digested with <t>BsrGI</t> and XbaI followed by hybridization with 5′-UTR specific probe. ( e ) Northern analysis for RNA isolated from Pb WT and Pb ASKO parasites indicating the absence of AS mRNA (1.75 kb) in Pb ASKO. For control, GAPDH (1.01 kb; lower panel) was used (full blots are shown in Supplementary Fig. 6 ). ( f ) Western blot analysis indicating the absence of AS (67 kDa) in Pb ASKO parasite lysate ( Supplementary Fig. 6 ). Parasite lysates of Pb WT and Pb ASKO containing 100 μg of total protein were used. For control, hsp60 (60 kDa) was used (lower panel). ( g ) Enzyme assays for Pb WT and Pb ASKO parasites using [U- 14 C]-aspartate. 200 μg of total protein was used per assay.
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    Generation of AS knockout in P. berghei . ( a ) Double cross-over recombination strategy followed to generate Pb ASKO. ( b ) PCR analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: PCR with Pb AS-specific primers; Lane 2 and 4: PCR with Pb GAPDH-specific primers (control); Lane M: 1 kb DNA ladder. ( c ) RT-PCR analysis for RNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: RT-PCR with Pb AS-specific primers; Lane 2 and 4: RT-PCR with Pb GAPDH specific primers (control); Lane M: 1 kb DNA ladder. ( d ) Southern analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Transgenic plasmid (TP) was also included as control to rule out the presence of any episomes. Genomic DNA preparations and TP were digested with BsrGI and XbaI followed by hybridization with 5′-UTR specific probe. ( e ) Northern analysis for RNA isolated from Pb WT and Pb ASKO parasites indicating the absence of AS mRNA (1.75 kb) in Pb ASKO. For control, GAPDH (1.01 kb; lower panel) was used (full blots are shown in Supplementary Fig. 6 ). ( f ) Western blot analysis indicating the absence of AS (67 kDa) in Pb ASKO parasite lysate ( Supplementary Fig. 6 ). Parasite lysates of Pb WT and Pb ASKO containing 100 μg of total protein were used. For control, hsp60 (60 kDa) was used (lower panel). ( g ) Enzyme assays for Pb WT and Pb ASKO parasites using [U- 14 C]-aspartate. 200 μg of total protein was used per assay.

    Journal: Nature Communications

    Article Title: Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections

    doi: 10.1038/ncomms9775

    Figure Lengend Snippet: Generation of AS knockout in P. berghei . ( a ) Double cross-over recombination strategy followed to generate Pb ASKO. ( b ) PCR analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: PCR with Pb AS-specific primers; Lane 2 and 4: PCR with Pb GAPDH-specific primers (control); Lane M: 1 kb DNA ladder. ( c ) RT-PCR analysis for RNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: RT-PCR with Pb AS-specific primers; Lane 2 and 4: RT-PCR with Pb GAPDH specific primers (control); Lane M: 1 kb DNA ladder. ( d ) Southern analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Transgenic plasmid (TP) was also included as control to rule out the presence of any episomes. Genomic DNA preparations and TP were digested with BsrGI and XbaI followed by hybridization with 5′-UTR specific probe. ( e ) Northern analysis for RNA isolated from Pb WT and Pb ASKO parasites indicating the absence of AS mRNA (1.75 kb) in Pb ASKO. For control, GAPDH (1.01 kb; lower panel) was used (full blots are shown in Supplementary Fig. 6 ). ( f ) Western blot analysis indicating the absence of AS (67 kDa) in Pb ASKO parasite lysate ( Supplementary Fig. 6 ). Parasite lysates of Pb WT and Pb ASKO containing 100 μg of total protein were used. For control, hsp60 (60 kDa) was used (lower panel). ( g ) Enzyme assays for Pb WT and Pb ASKO parasites using [U- 14 C]-aspartate. 200 μg of total protein was used per assay.

    Article Snippet: For Southern analysis, genomic DNA preparations (10 μg) from Pb WT and Pb ASKO parasites were subjected to BsrGI and XbaI digestion followed by hybridization with 5′-UTR-specific probe that was synthesized using Klenow Fragment (New England Biolabs) with 5′-UTR PCR product as a template in the presence of 5 μCi [α-32 P]-dATP.

    Techniques: Knock-Out, Polymerase Chain Reaction, Isolation, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Plasmid Preparation, Hybridization, Northern Blot, Western Blot

    Comparison of cloned plasmids produced from an equimolar mixture of three DNA fragments of different sizes by RC and LC. Plasmids retrieved from transformants generated by RC and LC were digested with Bgl II ( A ) or Bsr GI ( B ), and then run on 0.7 or 1.2% agarose gels, respectively. Plasmids were linearized by Bgl II digestion, and inserts were excised from the vector (pSP73) by Bsr GI digestion. Because the resultant clones by RC contained recombination site sequences in the vector portion, the size of the RC plasmid became slightly larger than that of the LC plasmid even when it carried the same insert. In (B), contaminating E.coli chromosomal DNA appeared as the uppermost band in both lanes and the excised βGal fragment, which should run slightly slower than the vectors, was not seen in either lane.

    Journal: Nucleic Acids Research

    Article Title: Directional cDNA library construction assisted by the in vitro recombination reaction

    doi:

    Figure Lengend Snippet: Comparison of cloned plasmids produced from an equimolar mixture of three DNA fragments of different sizes by RC and LC. Plasmids retrieved from transformants generated by RC and LC were digested with Bgl II ( A ) or Bsr GI ( B ), and then run on 0.7 or 1.2% agarose gels, respectively. Plasmids were linearized by Bgl II digestion, and inserts were excised from the vector (pSP73) by Bsr GI digestion. Because the resultant clones by RC contained recombination site sequences in the vector portion, the size of the RC plasmid became slightly larger than that of the LC plasmid even when it carried the same insert. In (B), contaminating E.coli chromosomal DNA appeared as the uppermost band in both lanes and the excised βGal fragment, which should run slightly slower than the vectors, was not seen in either lane.

    Article Snippet: MCS, Tet and βGal thus obtained were digested with Bsr GI (New England BioLabs), which cleaves at the TGTACA sequence within the common 15 bp core region of each att sequence, leaving four-base 5′ overhangs.

    Techniques: Clone Assay, Produced, Generated, Plasmid Preparation

    Assay of potential Tre-related off-target effects. (A) Nucleotide sequences of genomic sites and their locations in the human genome (in brackets). Sequences are aligned to the Tre recognition site loxLTR. Nucleotides that differ from loxLTR are shown in red. (B) Representation of the recombination assay in E. coli . and in HeLa cells, respectively. The evolution vector pEVO-Tre-target contains two directly repeated recombinase target sites (loxLTR) or the sequences GS1, GS2, GS3, GS4, GS5, GS6, and GS7. In E. coli , Tre is expressed from the P BAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC, and a chloramphenicol resistance marker (Cm r ). Recombination at the target sites leads to deletion of the 700 bp intervening region. Locations of the PCR primer binding sites (F, R) for detection of recombination are indicated. (C) Agarose gel showing the activity of Tre on loxLTR and the lack thereof for the seven genomic sites GS1 to GS7 (lanes 3–9). Upper panel: Recombination assayed in E. Coli . BsrG I/Xba I restriction digest results in a 4.9 kb fragment for non-recombined plasmid (two triangles) and a 4.2 kb fragment for recombined product (one triangle). Recombination tests on loxLTR served as negative and positive control (lanes 1 and 2). −, non-induced; +, induced with 1 mg/ml L-arabinose; M, DNA marker lane. Lower panel: Recombination assayed in HeLa cells. PCR using primers F and R that anneal to the vector DNA results in a 0.4 kb product when recombination occurs, while the non-recombined template results in a 1.1 kb PCR product. −, cotransfection with pIRESneo (i.e. no Tre expression); +, cotransfection with pIRESneo-Tre [18] .

    Journal: PLoS Pathogens

    Article Title: Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice

    doi: 10.1371/journal.ppat.1003587

    Figure Lengend Snippet: Assay of potential Tre-related off-target effects. (A) Nucleotide sequences of genomic sites and their locations in the human genome (in brackets). Sequences are aligned to the Tre recognition site loxLTR. Nucleotides that differ from loxLTR are shown in red. (B) Representation of the recombination assay in E. coli . and in HeLa cells, respectively. The evolution vector pEVO-Tre-target contains two directly repeated recombinase target sites (loxLTR) or the sequences GS1, GS2, GS3, GS4, GS5, GS6, and GS7. In E. coli , Tre is expressed from the P BAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC, and a chloramphenicol resistance marker (Cm r ). Recombination at the target sites leads to deletion of the 700 bp intervening region. Locations of the PCR primer binding sites (F, R) for detection of recombination are indicated. (C) Agarose gel showing the activity of Tre on loxLTR and the lack thereof for the seven genomic sites GS1 to GS7 (lanes 3–9). Upper panel: Recombination assayed in E. Coli . BsrG I/Xba I restriction digest results in a 4.9 kb fragment for non-recombined plasmid (two triangles) and a 4.2 kb fragment for recombined product (one triangle). Recombination tests on loxLTR served as negative and positive control (lanes 1 and 2). −, non-induced; +, induced with 1 mg/ml L-arabinose; M, DNA marker lane. Lower panel: Recombination assayed in HeLa cells. PCR using primers F and R that anneal to the vector DNA results in a 0.4 kb product when recombination occurs, while the non-recombined template results in a 1.1 kb PCR product. −, cotransfection with pIRESneo (i.e. no Tre expression); +, cotransfection with pIRESneo-Tre [18] .

    Article Snippet: Plasmid DNA was isolated from overnight cultures and digested with BsrG I and Xba I (NEB), resulting in different fragment sizes for recombined versus non-recombined substrate on agarose gels.

    Techniques: Recombination Assay, Plasmid Preparation, Marker, Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis, Activity Assay, Positive Control, Cotransfection, Expressing