Mega kit

Manufactured by Qiagen

Sourced in United Kingdom

The Qiagen Mega Kit is a laboratory equipment product designed for high-throughput nucleic acid purification. It offers efficient extraction and purification of DNA, RNA, or other biomolecules from a variety of sample types. The kit provides a streamlined workflow and reliable results for researchers and scientists working in various fields, such as genomics, molecular biology, and diagnostics.

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Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)

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EndoFree Plasmid Mega Kit (93 citations) Plasmid Mega Kit (55 citations) EndoFree Mega kit (7 citations) Mega preparation kit (4 citations) Endotoxin-Free Mega kit (3 citations) Plasmid Mega Prep Kit (3 citations) UltraClean Mega Prep soil DNA kit (2 citations) Mega DNA prep kit (2 citations) Endo-Free Mega Prep kit (2 citations) Qiagen mega plasmid purification kit (2 citations)

6 protocols using mega kit

Plasmid Amplification and Purification

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The reporter plasmids pCMVβ and pEGFP-N1 were amplified and purified from previously transformed stocks of DH5α Escherichia coli using antibiotic selective conditions and a Qiagen Mega Kit (Qiagen Ltd., Crawley, UK), [3] .
The pIRES-TdTomato plasmid was obtained from Clontech. Plasmid propagation and purification was performed using a Qiagen Mega Kit (pCMVβ and pEGFP-N1) or Qiagen HiSpeed Midi Kit (pIRES-TdTomato plasmid) (Qiagen Ltd., Crawley, UK).

Dul M., Stefanidou M., Porta P., Serve J., O'Mahony C., Malissen B., Henri S., Levin Y., Kochba E., Wong F.S., Dayan C., Coulman S.A, & Birchall J.C. (2017). Hydrodynamic gene delivery in human skin using a hollow microneedle device. Journal of controlled release : official journal of the Controlled Release Society, 265.

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Murine Carcinoma Vaccine Study

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Positive clones, unreactive to control sera from parental mice, were purified to monoclonality and converted to pBK-CMV phagemid by in vivo excision using XLOLR cells and ExAssist helper phage (Stratagene, LaJolla CA). Plasmid DNA was prepared using a Qiagen Mega kit (Qiagen, Valencia CA). The pBK-CMV3 plasmid with or without the indicated antigens was suspended in 50 μl of PBS with CFA/IFA and 50μg was given by intradermal injection into the ear. For implant studies, five mice per group for the early stage antigens and three mice per group for the late stage tumor antigens were vaccinated three times approximately 14 days apart. A syngeneic MMC mouse carcinoma line derived from a spontaneous tumor in a TgMMTV-neu mouse was implanted 1 week after the last vaccination. The MMC cell line was harvested using 2 mmol/L EDTA in PBS and washed with PBS before injection. Mice were inoculated with 3×10⁵ MMC cells subcutaneously on the mid-dorsum with a 23-gauge needle. For both studies, tumors were measured 3x/week with Vernier calipers and tumor volume was calculated as the product of length × width × height × π/6. Mice were euthanized when the tumor ulcerated, was >1000 mm³, or if the animal was ~1 year old. All mice were euthanized when the control mice developed clinically significant tumors.

Stanton S.E., Gad E., Corulli L.R., Lu H, & Disis M.L. (2019). Tumor-associated antigens identified early in mouse mammary tumor development can be effective vaccine targets.. Vaccine, 37(27), 3552-3561.

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Plasmid-Mediated VEGF Gene Delivery

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We used a eukaryotic expression vector encoding the VEGF165 gene for treatment [16 (link), 17 (link)]. The preparation and purification of the plasmid from phVEGF165-transformed Escherichia coli cultures were performed with the endotoxin-free column method (Qiagen Mega Kit, Qiagen Inc., Valencia, California). The purified plasmid was stored in vials and pooled for quality-control analysis. Aliquots of 2000 µg of phVEGF165 were diluted in a sterile saline to the volume of 10 ml.
10 ml of the solution containing 2 mg of VEGF165 plasmid gene was added to the MNC concentrate and incubated for 2 h before administration.
In RT-PCR stem cell tests the presence of VEGF165 mRNA was detected at a similar level before and after incubation with plasmid. These results prove a low rate of stem cell transfection by naked VEGF plasmid. However, based on our own data and literature data, we expected the occurrence of transfection of connective tissue cells at the place of injection of VEGF plasmid [23 (link), 24 ]. The expected effect would be an increase in the production of VEGF by ischemic tissue cells, which would significantly improve the settlement of the provided stem cells and their transformation into angioblast cells.

Skóra J., Pupka A., Janczak D., Barć P., Dawiskiba T., Korta K., Baczyńska D., Mastalerz-Migas A, & Garcarek J. (2014). Combined autologous bone marrow mononuclear cell and gene therapy as the last resort for patients with critical limb ischemia. Archives of Medical Science : AMS, 11(2), 325-331.

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Purification and Preparation of pEGFP

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The 4.7-kbp pEGFP-C1 (Roche Applied Science, Switzerland) was amplified in Escherichia coli and purified using the Endo-free plasmid mega-kit (QIAGEN Mega kit CA, USA). The purity of pEGFP was evaluated by UV spectroscopy (260/E280 nm ratio) and the concentration of pEGFP was measured using NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The measured concentration was approximately 500 ng/μl. Two types of pEGFP were used in accordance with the method of analysis. For electrophoresis experiments, normal pEGFP was heated at 94°C for 180 sec and then gradually cooled at room temperature for about 30 minutes. For AFM imaging experiments, normal pEGFP without heating was used.

Park D., Jung B.K., Park H., Lee H., Lee G., Park J., Shin U., Won J.H., Jo Y.J., Chang J.W., Lee S., Yoon D., Seo J, & Kim C.W. (2015). Sound Packing DNA: packing open circular DNA with low-intensity ultrasound. Scientific Reports, 5, 9846.

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Construction of Tf1-neo Plasmid Libraries

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Plasmids pHL2763 and pHL2770 contain WT and chromodomain deleted versions of Tf1-neo, respectively. They both contain a unique nucleotide tag in the U5 of the upstream LTR of Tf1-neo that distinguishes newly integrated copies from the pre-existing elements (4 (link)). The serial number plasmid libraries pHL2944 (WT) and pHL2943 (CHD) were constructed by ligating a PCR-amplified fragment of 250 bp of the upstream LTR into the unique XhoI and SpeI sites of pHL2763 and pHL2770. The 250 bp fragment was amplified from pHL2763 using the oligos HL2827 and HL2828 (See Supplementary Figure S1 for sequences of oligos). HL2827 included the unique SpeI sequence and a stretch of 8 nt randomized sequence and HL2828 had the unique XhoI sequence. The ligated products were electroporated into ElectroMAX DH5a cells (Invitrogen). Approximately 250 000 and 100 000 cfu(s) were pooled together and constituted the WT Tf1_s-neo and the CHD Tf1_s-CHD-neo libraries, respectively. Plasmid DNA was extracted using the Mega kit (Qiagen).

Chatterjee A.G., Esnault C., Guo Y., Hung S., McQueen P.G, & Levin H.L. (2014). Serial number tagging reveals a prominent sequence preference of retrotransposon integration. Nucleic Acids Research, 42(13), 8449-8460.

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Hammerhead Ribozyme Crystallization and Kinetics

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The two trans-acting versions of the hammerhead ribozyme were designed based on the previously reported sequences of the SELEX derived RzB^{41 (link)} and the S. mansoni ribozyme.^{24 (link)} To make the ribozyme strand, the ribozyme sequence was placed between a T7 promotor and an EarI restriction site, which was then inserted into pUC-19 and amplified in Escherichia coli DH5α cells. DNA plasmid was obtained using QIAGEN Mega kit. In vitro transcription and RNA purification by denaturing polyacrylamide gel electrophoresis (PAGE) were performed as previously described.⁴³ The RNA sample was concentrated using an Amicon Ultra centrifugal filter and stored at −20 °C.
For crystallization, a 20-nt inhibitor strand carrying a 2′-deoxy modification at the C17 position was purchased from Thermo Scientific, deprotected according to manufacturer's guidelines, and further purified by denaturing PAGE gel. For solution kinetic experiments, an all ribose substrate used was chemically synthesized and DY-547 labeled at its 5′-end.

Mir A., Chen J., Robinson K., Lendy E., Goodman J., Neau D, & Golden B.L. (2015). Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction. Biochemistry, 54(41), 6369-6381.

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