r3140  (New England Biolabs)

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

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

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

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