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Complementary oligos

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Complementary oligos are synthetic DNA or RNA sequences designed to bind to a specific target sequence. They are used in various molecular biology applications such as gene expression analysis, PCR, and hybridization techniques.

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Lab products found in correlation

Stellar competent e coli cells, by Takara Bio (2 mentions) Zymopure maxiprep kit, by Zymo Research (2 mentions) Storage phosphor screen, by GE Healthcare (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Lenticrispr v2, by Addgene (1 mentions) Polybrene, by Merck Group (1 mentions) Puromycin, by InvivoGen (1 mentions)

5 protocols using complementary oligos

1

Cloning Cas9/gRNA Constructs for CRISPR Experiments

Cited 2 times
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All CRISPR gRNAs used in SGE and essentiality experiments were cloned into pX45924 (link). This plasmid expresses the gRNA from a U6 promoter, as well as a Cas9–2A-puromycin resistance (-puroR) cassette. S. pyogenes Cas9 target sites were chosen for SGE experiments on multiple criteria, assessed in the following order: 1.) To induce cleavage within BRCA1 coding sequence, 2.) To target a genomic site permissive to synonymous substitution within the guanine dinucleotide of the PAM or the protospacer, 3.) To have minimal predicted off-target activity47 (link), 4.) To have maximal predicted on-target activity48 (link).
Complementary oligos ordered from Integrated DNA Technologies (IDT) were annealed, phosphorylated, diluted and ligated into BbsI-digested and gel-purified pX459, as described24 (link). Ligation reactions were transformed into E. coli (Stellar competent cells, Takara), which were plated on ampicillin. Colonies were cultured and Sanger sequenced to confirm correct gRNA sequences. Purification of sequence-verified plasmids for transfection was performed with the ZymoPure Maxiprep kit (ZymoResearch). For targeting LIG4 in HAP1 cells, pX45824 (link) was used instead of pX459, which expresses EGFP in lieu of puroR.
Findlay G.M., Daza R.M., Martin B., Zhang M.D., Leith A.P., Gasperini M., Janizek J.D., Huang X., Starita L.M, & Shendure J. (2018). Accurate classification of BRCA1 variants with saturation genome editing. Nature, 562(7726), 217-222.
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2

8-oxoG Cleavage Assay Protocol

Cited 1 time
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We obtained 31-mer oligos containing 8-oxoG at position X and complementary oligos with a C opposite X from Integrated DNA Technologies (Coralville, IA). The oligo sequence was 5′-GTGACTACGAGACCTXATGTGACTGAGAGAG- 3′, as previously described [105] (link). Cleavage reactions with bacterially purified proteins were conducted using 50 nM of proteins and 0.08 U of human OGG1 (New England Biolabs) in 25 mM NaCl, 10 mM Tris (pH 7.5), 1 mM MgCl2, 5 mM EDTA (pH 8.0), 5% glycerol, 1 mM of DTT, and 1 pmol of 32P radiolabeled double-stranded oligonucleotides containing an 8-oxoG base (Figure S5). Reactions with total cell extracts were performed as described by [106] (link). In both assays, cleavage reactions were performed at 37°C as previously described. The DNA was loaded on a prewarmed 20% polyacrylamide-urea gel (19∶1) and separated by electrophoresis in Tris-borate and EDTA (TBE; pH 8.0) at constant 20 mAmp. The radiolabeled DNA fragments were visualized by storage phosphor screen (GE Healthcare).
Ramdzan Z.M., Vadnais C., Pal R., Vandal G., Cadieux C., Leduy L., Davoudi S., Hulea L., Yao L., Karnezis A.N., Paquet M., Dankort D, & Nepveu A. (2014). RAS Transformation Requires CUX1-Dependent Repair of Oxidative DNA Damage. PLoS Biology, 12(3), e1001807.
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3

Cloning Strategy for SPA4CT Sequence

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A cloning strategy was designed and implemented to generate the SPA4CT sequence (25 (link)). pLVX APP-770 was digested with EcoRI and NotI to generate a 366 base pair terminal APP insert. Complementary oligos (purchased from Integrated DNA Technologies) were annealed to form an insert with XhoI and EcoRI overhangs. pLVX was digested with XhoI and NotI and religated together with the two inserts. For specific sequences, see Supplementary Table S4. SPA4CT-T43P was generated using the Q5 Site-Directed Mutagenesis Kit (NEB E0554S) using pLVX SPA4CT as a template. For specific sequences of primers and inserts, please see Supplementary Table S4.
Kleffman K., Levinson G., Rose I.V., Blumenberg L.M., Shadaloey S.A., Dhabaria A., Wong E., Galán-Echevarría F., Karz A., Argibay D., Von Itter R., Floristán A., Baptiste G., Eskow N.M., Tranos J.A., Chen J., Vega y Saenz de Miera E.C., Call M., Rogers R., Jour G., Wadghiri Y.Z., Osman I., Li Y.M., Mathews P., DeMattos R., Ueberheide B., Ruggles K.V., Liddelow S.A., Schneider R.J, & Hernando E. (2022). Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis. Cancer discovery, 12(5), 1314-1335.
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4

Cloning Cas9/gRNA Constructs for CRISPR Experiments

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
All CRISPR gRNAs used in SGE and essentiality experiments were cloned into pX45924 (link). This plasmid expresses the gRNA from a U6 promoter, as well as a Cas9–2A-puromycin resistance (-puroR) cassette. S. pyogenes Cas9 target sites were chosen for SGE experiments on multiple criteria, assessed in the following order: 1.) To induce cleavage within BRCA1 coding sequence, 2.) To target a genomic site permissive to synonymous substitution within the guanine dinucleotide of the PAM or the protospacer, 3.) To have minimal predicted off-target activity47 (link), 4.) To have maximal predicted on-target activity48 (link).
Complementary oligos ordered from Integrated DNA Technologies (IDT) were annealed, phosphorylated, diluted and ligated into BbsI-digested and gel-purified pX459, as described24 (link). Ligation reactions were transformed into E. coli (Stellar competent cells, Takara), which were plated on ampicillin. Colonies were cultured and Sanger sequenced to confirm correct gRNA sequences. Purification of sequence-verified plasmids for transfection was performed with the ZymoPure Maxiprep kit (ZymoResearch). For targeting LIG4 in HAP1 cells, pX45824 (link) was used instead of pX459, which expresses EGFP in lieu of puroR.
Findlay G.M., Daza R.M., Martin B., Zhang M.D., Leith A.P., Gasperini M., Janizek J.D., Huang X., Starita L.M, & Shendure J. (2018). Accurate classification of BRCA1 variants with saturation genome editing. Nature, 562(7726), 217-222.
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5

Lentiviral CRISPR-Cas9 Knockdown of MTA1

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The online guide design tool from DNA 2.0 gRNA (now known as ATUM, Newark, CA) was used to identify sgRNAs. The DNA sequence corresponding to the annotated stem loop miRNA (www.miRBase.org) was used as an input sequence. The highest scoring guides and/or those closest to the mature miRNA sequence were selected. Complementary oligos containing the sgRNA sequence and BsmBI overhangs were synthesized (Integrated DNA Technologies), annealed, digested with BsmBI and ligated into the lentiCRISPR v2, a gift from Feng Zhang (Addgene, # 52961) [20 (link)]. MTA1-sgRNA-1: 5′- CTCCAAGGCCATCTCGGCGC-3′; MTA1-sgRNA-3: 5′- CAGCTGCGGCGCTCATGTGC-3 and MTA1-sgRNA-5: 5’-CTCTGTGGGCACCTTCGCAC-3′. MCF7 and MDA-MB-231 cells were infected with lentivirus in the presence of 8 μg/ml polybrene (Sigma-Aldrich). Approximately 48 h post-infection cells were selected by treating with 1 μg/ml puromycin (InvivoGen, San Diego, CA) for 3 days.
Hannafon B.N., Gin A.L., Xu Y.F., Bruns M., Calloway C.L, & Ding W.Q. (2019). Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells. Cell Communication and Signaling : CCS, 17, 13.
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