xho i  (New England Biolabs)

Journal: PeerJ

Article Title: A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

doi: 10.7717/peerj.8491

Figure Lengend Snippet: Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.

Article Snippet: The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, 1 ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH2 O to 10 ul.

Techniques: Expressing, Clone Assay, Amplification, Polymerase Chain Reaction, Incubation

Journal: Nature Communications

Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

doi: 10.1038/s41467-019-13719-9

Figure Lengend Snippet: Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

Article Snippet: Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol.

Techniques: Isolation, Mutagenesis, Amplification, Produced, Sequencing

Journal: Nature Communications

Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

doi: 10.1038/s41467-019-13719-9

Figure Lengend Snippet: Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

Article Snippet: Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol.

Techniques: Whole Genome Amplification, Sequencing, Amplification, Ligation

Journal: PLoS ONE

Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

doi: 10.1371/journal.pone.0114208

Figure Lengend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

Article Snippet: Portions of lysates were further heat inactivated (HI) at 95°C for 10 min. One µg of the commercial plasmid pECFP-N1 (Clonetech, CA, USA) was subjected to either MS11 P+ lysate or HI lysate together with CutSmart buffer (New England Biolabs, Ipswich, MA, USA) for 1 h. As controls, circular/uncut pECFP-N1 was used as well as HindIII (Roche, Mannheim, Germany) linearized pECFP-N1.

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation

Journal: Journal of Virology

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

doi: 10.1128/JVI.00539-17

Figure Lengend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

Article Snippet: For exonuclease T5 (Exo T5) digestion, 20 μl PF DNA sample was treated with 0.5 μl (5 units) Exo T5 in 1× Cutsmart buffer (NEB) in a total volume of 23 μl ( ) at 37°C for 2 to 3 h. Phenol extraction was then used to remove the nucleases before qPCR.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

Journal: Journal of Virology

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

doi: 10.1128/JVI.00539-17

Figure Lengend Snippet: Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

Article Snippet: For exonuclease T5 (Exo T5) digestion, 20 μl PF DNA sample was treated with 0.5 μl (5 units) Exo T5 in 1× Cutsmart buffer (NEB) in a total volume of 23 μl ( ) at 37°C for 2 to 3 h. Phenol extraction was then used to remove the nucleases before qPCR.

Techniques: Countercurrent Chromatography, Modification, Agarose Gel Electrophoresis, Southern Blot, Migration

Journal: Journal of Virology

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

doi: 10.1128/JVI.00539-17

Figure Lengend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

Article Snippet: For exonuclease T5 (Exo T5) digestion, 20 μl PF DNA sample was treated with 0.5 μl (5 units) Exo T5 in 1× Cutsmart buffer (NEB) in a total volume of 23 μl ( ) at 37°C for 2 to 3 h. Phenol extraction was then used to remove the nucleases before qPCR.

Techniques: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker