Hispeed maxiprep kit, by Qiagen - Product details

1

Tol2-based Zebrafish Transgenic Constructs

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An 4.1 kb mitfa promoter fragment, the coding sequences for EGFP and bovine TWIST2, and the IRES casette were amplified separately and engineered into a vector containing Tol2 sites (pTKXIGΔin; Kawakami Laboratory) using the Gibson Assembly^® Cloning kit from NEB. We thus generated the plasmids pmitfa_EGFP (control construct without TWIST2 expression cassette) and pmitfa_btaTWIST2_EGFP. A detailed map of these plasmids is given in S2 and S3 Figs.
An expression casette containing the gamma-crystallin promoter and the coding sequence of CFP was taken from Tg(hsp70l::caALK5) [43 (link)] and inserted into pmitfa_EGFP and and pmitfa_btaTWIST2_EGFP by Gibson cloning to generate pmitfa_EGFP_cryst_CFP and pmitfa_TWIST2_EGFP_cryst_CFP. A detailed map of these plasmids is given in S4 and S5 Figs. Plasmid DNA was purified using the Qiagen HiSpeed Maxiprep kit and the correct sequence was verified by Sanger sequencing.

Awasthi Mishra N., Drögemüller C., Jagannathan V., Keller I., Wüthrich D., Bruggmann R., Beck J., Schütz E., Brenig B., Demmel S., Moser S., Signer-Hasler H., Pieńkowska-Schelling A., Schelling C., Sande M., Rongen R., Rieder S., Kelsh R.N., Mercader N, & Leeb T. (2017). A structural variant in the 5’-flanking region of the TWIST2 gene affects melanocyte development in belted cattle. PLoS ONE, 12(6), e0180170.

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2

IFITM3 Knockout and Overexpression in U138 Cells

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CRISPR constructs were purchased from the RNAi core at Memorial Sloan Kettering Cancer Center and purified with the HiSpeed Maxiprep kit (Qiagen). U138 cells were transfected using Lipofectamine LTX according to the manufacturer’s instructions with px459 vector containing Cas9 and guide RNAs (either an empty vector or one of three IFITM3 guides). Transfected cells were incubated for 48 hrs prior to puromycin selection. Single cells were extracted and expanded. IFITM3 KO was confirmed by sequencing and WB.
Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector. U138 EV and U138 IFITM3−/− cells were transfected with either pcDNA3.1 empty vector of pcDNA3.1 IFITM3 construct using Lipofectamine LTX according to the manufacturer’s instructions and allowed to incubate for 48 hrs prior to membrane preparation.

Hur J.Y., Frost G.R., Wu X., Crump C., Pan S.J., Wong E., Barros M., Li T., Nie P., Zhai Y., Wang J.C., TCW J., Guo L., McKenzie A., Ming C., Zhou X., Wang M., Sagi Y., Renton A.E., Esposito B.T., Kim Y., Sadleir K.R., Trinh I., Rissman R.A., Vassar R., Zhang B., Johnson D.S., Masliah E., Greengard P., Goate A, & Li Y.M. (2020). Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease. Nature, 586(7831), 735-740.

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3

IFITM3 Knockout and Overexpression in U138 Cells

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CRISPR constructs were purchased from the RNAi core at Memorial Sloan Kettering Cancer Center and purified with the HiSpeed Maxiprep kit (Qiagen). U138 cells were transfected using Lipofectamine LTX according to the manufacturer’s instructions with px459 vector containing Cas9 and guide RNAs (either an empty vector or one of three IFITM3 guides). Transfected cells were incubated for 48 hrs prior to puromycin selection. Single cells were extracted and expanded. IFITM3 KO was confirmed by sequencing and WB.
Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector. U138 EV and U138 IFITM3−/− cells were transfected with either pcDNA3.1 empty vector of pcDNA3.1 IFITM3 construct using Lipofectamine LTX according to the manufacturer’s instructions and allowed to incubate for 48 hrs prior to membrane preparation.

Hur J.Y., Frost G.R., Wu X., Crump C., Pan S.J., Wong E., Barros M., Li T., Nie P., Zhai Y., Wang J.C., TCW J., Guo L., McKenzie A., Ming C., Zhou X., Wang M., Sagi Y., Renton A.E., Esposito B.T., Kim Y., Sadleir K.R., Trinh I., Rissman R.A., Vassar R., Zhang B., Johnson D.S., Masliah E., Greengard P., Goate A, & Li Y.M. (2020). Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease. Nature, 586(7831), 735-740.

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4

Robust Plasmid DNA Purification Protocol

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pMC454 was
transformed into LZ54 cells via heat shock, conditioning,
and growth at 30 °C as previously described.^{24 (link)} A 10 mL overnight culture grown in LB/Amp at 30 °C
was used to inoculate 350 mL of modified TB (12 g of tryptone, 48
g of yeast extract, 30 mL of glycerol, 0.1 mL of antifoam 204, 2.32
g of KH₂PO₄, and 12.54 g of K₂HPO₄ per liter). Inoculated medium was incubated
at 30 °C while being shaken at 225 rpm to an OD₆₀₀ of 2.0. The culture was then heat shocked to induce λ-integrase
activity via the addition of 350 mL of modified TB preheated to 60
°C, and cultures were shaken at 42 °C for 30 min before
norfloxacin was added to a final concentration of 30 μg/mL.
The temperature was returned to 30 °C, and cells were shaken
for an additional 1 h.^{24 (link)}Cells were
harvested via centrifugation at 6000g for 15 min,
resuspended in Buffer P1 (Qiagen), and frozen at −20
°C. Plasmid DNA was purified using a HiSpeed Maxiprep
Kit (Qiagen) according to the manufacturer’s instructions and
eluted into 1 mL of buffer EB [10 mM Tris-HCl (pH 8.5)]. To confirm
that the λ-integrase disintegration reaction and decatenation
were complete, 1 μL of eluent was run on a 1% agarose gel (Figure 1).

Anderson B.G, & Stivers J.T. (2014). Variola Type IB DNA Topoisomerase: DNA Binding and Supercoil Unwinding Using Engineered DNA Minicircles. Biochemistry, 53(26), 4302-4315.

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5

Establishing Stable GFP-Expressing HT1080 Cells

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Plasmid DNA was extracted from 150 mL of DH10β culture using the Qiagen HiSpeed Maxi Prep kit, digested at 37°C, and purified by phenol-chloroform extraction. To establish stable, green fluorescent protein (GFP) expressing cells, three batches of 12 x 10 6 viable cells were re-suspended in RPMI-1640 media (0.75 mL per cuvette) and transfected with 50 μg of pIRES-hrGFP DNA using a 4 mm electroporation cuvette and Gene Pulser XCell (BioRad) at 350 V and 950 μF of capacitance. Transfection efficiencies were typically 80%. Cells were transferred to CD 50/50 media and recovered 48 hours before selection pressure was applied.
Concentrations of 50, 100 and 250 μg/ml of Zeocin were used to establish the HT1080 libraries.
Selective pressure was maintained until culture viability was above 90%.

Cheng J.K., Lewis A.M., Kim do S., Dyess T, & Alper H.S. (2016). Identifying and retargeting transcriptional hot spots in the human genome. Biotechnology journal, 11(8).

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6

Engineered FAK and SrcY530F constructs

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pAcGFP1-Hyg-C1-FAK (human wild-type FAK fused to Cter of GFP) was constructed by inserting the FAK PCR amplified insert in the BglII/SalI restriction sites of pAcGFP1-Hyg-C1 (Clontech). The I936E-I998E-FAK (FAK^I936/I998) double mutant was generated using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). Briefly, pAcGFP1-Hyg-C1-FAK was used as a template to generate the FAK^I936/I998construct using FAKI936E-S (5′-GCCTGGTGAAAGCTGTCGAGGAGATGTCCAGTAAAATCCAGC-3′), FAKI936E-AS (5′-GCTGGATTTTACTGGACATCTCCTCGACAGCTTTCACCAGGC-3′), FAKI998E-S (5′-GAACTCTGACCTGGGTGAGCTCGAAAACAAGATGAAACTGGCC-3′), and FAKI998E-AS (5′-GGCCAGTTTCATCTTGTTTTCGAGCTCACCCAGGTCAGAGTTC-3′) primers. pcDNA3.1-mCherry-SrcY530F was generated by inserting the PCR product mCherry-SrcY530F in pcDNA3.1(zeo) (Invitrogen). All constructs were amplified and purified using Qiagen Hispeed Maxiprep kits and specific point mutations were verified by sequencing.

Deramaudt T.B., Dujardin D., Noulet F., Martin S., Vauchelles R., Takeda K, & Rondé P. (2014). Altering FAK-Paxillin Interactions Reduces Adhesion, Migration and Invasion Processes. PLoS ONE, 9(3), e92059.

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7

Brunello CRISPR Library Amplification

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The Brunello CRISPR library targeting the human genome was obtained from Addgene (via John Doench and David Root, Addgene #73178), and 400ng was electroporated into Stbl4 bacteria (Thermo Fisher). Colonies were grown by incubation at 30°C on large bioassay plates; bacteria were harvested by scraping colonies in cold LB medium. DNA was purified using HiSpeed Maxi prep kits (Qiagen).

Dersh D., Phelan J.D., Gumina M.E., Wang B., Arbuckle J.H., Holly J., Kishton R.J., Markowitz T.E., Seedhom M.O., Fridlyand N., Wright G.W., Huang D.W., Ceribelli M., Thomas C.J., Lack J.B., Restifo N.P., Kristie T.M., Staudt L.M, & Yewdell J.W. (2020). Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas. Immunity, 54(1), 116-131.e10.

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8

Plasmid Fusion Protein Expression

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The plasmids encoding for the following fusion proteins were kindly provided by the following researchers: mCherry-Galactosyltransferase (mCherry-GT) (Miller et al., 2009) was provided by I. Kaverina (Vanderbilt University Medical Center, Nashville, USA), 2Nacetylglucosaminyltransferase I (NAGFP) (Shima et al., 1997) was a gift from D. Shima and G. Warren (Imperial Cancer Research Fund, London, UK), dynamitin-EGFP (Dohner et al., 2002) is from the laboratory of R. Vallee (Columbia University, NY, USA), and mCherryvinculin (Wolfenson et al., 2009) was provided by I. Lavelin (Weizmann Institute of Science, Rehovot, Israel). Mouse EGFP-tagged dynein intermediate chain IC2C (DIC) was obtained from Addgene (King et al., 2003) . Human wild-type FAK fused to C-terminus of EGFP or mCherry are described in (Deramaudt et al., 2014) . FAK-EGFP plasmid was used as template to generate the S732A and S732D substitution constructs with the QuikChange II XL sitedirected mutagenesis kit (Agilent Technologies). The following sequences were used to design the primers used for the PCR reactions: S732A: 5'-GCGAAGGATTTTATCCCGCTCCACAGCACATGGTAC-3'; S732D: 5'-GCGAAGGATTTTATCCCGACCCACAGCACATGGTAC-3'. All constructs were amplified and purified using Qiagen Hispeed Maxiprep kits and verified by sequencing.

Fructuoso M., Legrand M., Mousson A., Steffan T., Vauchelles R., De Mey J., Sick E., Rondé P, & Dujardin D. (2020). FAK regulates dynein localisation and cell polarity in migrating mouse fibroblasts. Biology of the cell, 112(2).

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Hispeed maxiprep kit

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8 protocols using hispeed maxiprep kit

Tol2-based Zebrafish Transgenic Constructs

IFITM3 Knockout and Overexpression in U138 Cells

IFITM3 Knockout and Overexpression in U138 Cells

Robust Plasmid DNA Purification Protocol

Establishing Stable GFP-Expressing HT1080 Cells

Engineered FAK and SrcY530F constructs

Brunello CRISPR Library Amplification

Plasmid Fusion Protein Expression

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