To examine the mutation rate in the mitochondrial DNA, a 431 bp fragment of the D-loop region was cloned using the InFusion HD cloning kit (Clontech) as follows. Total DNA templates including mtDNA were isolated from K512-vector and K512-EF1α-MajSAT cells using the QIAamp DNA Mini Kit (Qiagen), and PCR was performed with LA-Taq polymerase (TaKaRa) mixed with Pfu turbo DNA Polymerase (Stratagene). The primers used were as follows: Fw: 5′-CTGGACTAGTGGATCCTCTTTTTATTTTGGCCTACTTT-3′, Rv: 5′-TACCGAGCTCGGATCCCATCTAAGCATTTTCAGTGC-3′ (ref. 31 (link)). The pcDNA 3.1(−) vector was digested at the BamHI site and dephosphorylated with Shrimp Alkaline Phosphatase (TaKaRa) at 37 °C for 15 min. Amplified fragments and linearized vector were incubated with 5 × InFusion HD Enzyme premix at 50 °C for 15 min and subsequently transformed into ECOS Competent E. coli DH5α. After 16 h, at least twenty colonies from each sample were picked up, and cloned vectors were collected using the QIAprep 96 Turbo Miniprep Kit (Qiagen). The inserted D-loop sequences of the cloned libraries were determined using a T7 sequence primer (5′-TAA TAC GAC TCA CTA TAG GG-3′). The mutations were determined by comparison with the referee sequences (GenBank: NC_005089.1).
Qiaprep 96 turbo miniprep kit
Manufactured by Qiagen
Sourced in Germany
The QIAprep 96 Turbo Miniprep Kit is a laboratory equipment product designed for the rapid and efficient purification of high-quality plasmid DNA from bacterial cultures. It utilizes a 96-well format to enable parallel processing of multiple samples.
Automatically generated - may contain errors
Lab products found in correlation
Zeba plate, by Thermo Fisher Scientific (2 mentions)
Ni sepharose high performance, by GE Healthcare (2 mentions)
Labchip gxii, by PerkinElmer (2 mentions)
Shrimp alkaline phosphatase, by Takara Bio (1 mentions)
La taq polymerase, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Plasmid miniprep kit, by Qiagen (1 mentions)
Quant it dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Superscript system, by Thermo Fisher Scientific (1 mentions)
Fasttrack magmaxi mrna isolation kit, by Thermo Fisher Scientific (1 mentions)
Electromax dh10b competent cells, by Thermo Fisher Scientific (1 mentions)
Rneasy maxi, by Qiagen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Qiaquick 96 pcr purification kit, by Qiagen (1 mentions)
Primestar gxl, by Takara Bio (1 mentions)
Qpix2 colony picking robot, by Genetix (1 mentions)
Pcr2.1 topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using qiaprep 96 turbo miniprep kit
1
Cloning and Sequencing Mitochondrial D-loop
2
High-Throughput Antibody Variant Purification
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Example 2
The top leads from the initial round of screening were cultured and plasmids encoding the antibody genes of interest were isolated using a QIAprep 96 Turbo® Miniprep Kit (Qiagen) according to the manufacturer's instructions. DNA was added to 4 mL of cell-free reaction medium to achieve a final concentration of 10 μg/mL. The cell-free reaction medium was then incubated overnight for 12 hr at 30° C., at 650 rpm.
The expressed variants from clarified cell-free reactions were purified via immobilized metal ion affinity chromatography (IMAC) using a semi-automated high throughput batch purification method. Briefly, purifications were performed in a 96-well plate format where 50 μL/well of IMAC resin (Ni Sepharose® High Performance, GE Healthcare) was equilibrated in IMAC binding buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole), incubated with 1 mL cell-free reaction for 15 minutes, followed by two washes in IMAC binding buffer. His-tagged antibody variants were then eluted using 200 μL IMAC elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole), and buffer exchanged into PBS using a 96-well Zeba™ plate (7 kDa MWCO, Thermo Fisher). Purified antibodies were quantified via high throughput capillary electrophoresis using the LabChip GXII® (Perkin Elmer) against a trastuzumab standard curve, according to the manufacturer's instructions.
3
Barcode Quantification of Dub-seq Library
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Plasmid DNA from Dub-seq library samples was extracted either individually using the Plasmid miniprep kit (Qiagen) or in 96-well format with a QIAprep 96 Turbo miniprep kit (Qiagen). Plasmid DNA was quantified with the Quant-iT dsDNA BR assay kit (Invitrogen). The BarSeq PCR of UP barcodes was done as previously described13 (link) with ~50 ng of plasmid template per BarSeq PCR reaction. To quantify the reproducibility of both UP and DOWN barcodes in competitive growth experiments, we collected plasmid DNA from nickel and cobalt experiments, and amplified both UP and DOWN barcodes in two separate PCRs using the same plasmid library template. For BarSeq PCR of DOWN barcodes, we used universal-forward-primer DT_BarSeq_p1_FW and reverse primer DT_BarSeq_IT017. The PCR cycling conditions and purification steps were same as for the UP barcodes13 (link). All experiments done on the same day and sequenced on the same lane are considered as a “set”.
4
Screening cDNA Library with Hybridomas
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Total RNA was prepared from the murine CH27 cell line by RNeasy Maxi (QIAGEN Inc.) and was purified with FastTrack MAG Maxi mRNA isolation kit (Invitrogen) to obtain poly(A)+ RNA. cDNA was synthesized with the SuperScript system (Invitrogen) and was cloned into SPORT6 vector with SalI and NotI restriction sites. ElectroMAX DH10B competent cells (Invitrogen) were transformed by electroporation, and after titration, E. coli (~150 clones/well) were inoculated overnight into 96-well format culture blocks (10 blocks). Plasmids were purified with a Qiaprep 96 Turbo miniprep kit (QIAGEN) and were transfected to HEK293T cells with Lipofectamine 2000 (Invitrogen) in 96-well flat-bottom plates and left overnight. Hybridomas were cocultured with cDNA-transfected 293T cells for 24 h, after which mIL-2 amounts in the supernatants were obtained. Positive clones were selected for secondary and tertiary screenings. Subpool libraries (~20 clones/well, 48 wells) and clone libraries (1 clone/well, 96 wells) were prepared and screened. Positive clones were sequenced to identify the specificity of the transfected cDNA.
5
Automated Massive Plasmid Cloning in Sky BioFoundry
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Robot-assisted automated gene cloning in the SKy BioFoundry, a member of the Global Biofoundry Alliance, was used for massive plasmid cloning experiments (32 (link)). Briefly, high-fidelity polymerase (PrimeSTAR GXL, Takara Bio Inc., Shiga, Japan) was used for PCR. DNA fragments were amplified in an On Deck Thermal Cycler (INHECO, Germany) on the SKy BioFoundry platform. All PCR products and plasmids for the crRNA library were purified and extracted using the QIAprep 96 Turbo Miniprep Kit (Qiagen, Germany) and the QIAquick 96 PCR Purification Kit (Qiagen, Germany). The primers used for crRNA library construction are listed in Supplementary Table S3 . All oligonucleotides were purchased from Genotech (Daejeon, Korea), and DNA sequencing was performed by Macrogen (Seoul, Korea).
6
Streamlining Clonase Efficiency Tracking
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In cases where Clonase efficiency values were >66%, average of at least 2 hits out of 3 colonies screened, the entire liquid culture plate was replicated with a 96-pin replicator onto an agar plate with the same dimensions as a 96-well plate. The plate was immediately submitted to an outside vendor (Quintarabio, Berkeley, CA), and after growth overnight sequencing was performed on amplified DNA from each clone. If Clonase efficiency values were <66% (Taq and Pfu polymerase reactions), a rearray step was added, using a Qpix2 colony picking robot (Genetix) to maximize the number of clones with correct-size insert on one plate. For comparing sequencing results using cells versus miniprep DNA, one plate of colonies picked from a Taq cloning reaction was replicated into a 96-well deep well plate with 800 mL media per well and grown overnight with shaking at 300 rpm. Cells were pelleted, and DNA was prepared using a Qiaprep 96 Turbo Miniprep Kit (Qiagen). Eluted DNA was submitted directly for sequencing.
7
High-throughput Protein Purification Protocol
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Example 2
The top leads from the initial round of screening were cultured and plasmid minipreps were performed using a QIAprep® 96 Turbo miniprep kit (Qiagen) according to the manufacturer's instructions. 10 μg/mL miniprepped DNA was added to 4 mL cell-free reactions and incubated overnight for 12 hr at 30° C., at 650 rpm.
Expressed variants from clarified cell-free reactions were purified via immobilized metal ion affinity chromatography (IMAC) purification using a semi-automated high throughput batch purification method. Briefly, purifications were performed in a 96-well plate format where 50 μL/well of IMAC resin (Ni Sepharose High Performance, GE Healthcare) was equilibrated in IMAC binding buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole), incubated with 1 mL cell-free reaction for 15 minutes followed by two washes in IMAC binding buffer. His-tagged antibody variants were then eluted using 200 μL IMAC elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole) and buffer exchanged into PBS using a 96-well Zeba plate (7 kD MWCO, Thermo Fisher). Purified antibodies were quantified via high throughput capillary electrophoresis using the LabChip GXII (Perkin Elmer) against a Herceptin standard curve, according to the manufacturer's instructions.
8
cDNA Sequencing and Cloning Protocol
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