r3140  (New England Biolabs)

Journal: bioRxiv

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

doi: 10.1101/2020.05.20.105924

Figure Lengend 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.

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.

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

Journal: bioRxiv

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

doi: 10.1101/2020.05.20.105924

Figure Lengend 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.

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.

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

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: PeerJ

Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

doi: 10.7717/peerj.9841

Figure Lengend Snippet: Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.

Article Snippet: 3A assemblyA BioBrick-compatible plasmid that encodes chloramphenicol acetyltransferase, RP4 oriT-pUC57-mini-cat (2 µg) in 1× NEB CutSmart buffer (80 µL total reaction volume) by 20 units each of EcoRI-HF, PstI-HF and NotI-HF (so as to eliminate the sticky ends of its stuffer fragment), dephosphorylated in reactions with NEB Calf Intestinal Phosphatase.

Techniques: Plasmid Preparation, Expressing, Recombinant, Construct, Low Copy Number

Journal: PeerJ

Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

doi: 10.7717/peerj.9841

Figure Lengend Snippet: Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.

Article Snippet: 3A assemblyA BioBrick-compatible plasmid that encodes chloramphenicol acetyltransferase, RP4 oriT-pUC57-mini-cat (2 µg) in 1× NEB CutSmart buffer (80 µL total reaction volume) by 20 units each of EcoRI-HF, PstI-HF and NotI-HF (so as to eliminate the sticky ends of its stuffer fragment), dephosphorylated in reactions with NEB Calf Intestinal Phosphatase.

Techniques: Subcloning, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Stable Transfection, In Vivo, Construct, Selection

Journal: PeerJ

Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

doi: 10.7717/peerj.9841

Figure Lengend Snippet: 4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .

Article Snippet: 3A assemblyA BioBrick-compatible plasmid that encodes chloramphenicol acetyltransferase, RP4 oriT-pUC57-mini-cat (2 µg) in 1× NEB CutSmart buffer (80 µL total reaction volume) by 20 units each of EcoRI-HF, PstI-HF and NotI-HF (so as to eliminate the sticky ends of its stuffer fragment), dephosphorylated in reactions with NEB Calf Intestinal Phosphatase.

Techniques: Plasmid Preparation, Methylation, In Vivo, Ligation, Recombinant