Do not add bleach or acidic solutions directly to the preparation waste. Throughout the remainder of this document the supplied cell resuspension solution cra, cell lysis solution cla and neutralization solution nsb are referred to as cell resuspension solution, cell lysis. Hispeed plasmid purification handbook harvard university. Mega, giga purelink hipure filter maxi plasmid dna. A resource portal for protocols in bioscience including molecular biology, cell biology, immunology, plant biology, developmental biology, neuroscience, bioinformatics, genetics. Qiagen maxi prep kit low endotoxin without additional steps. The qiagen bench guide is designed to help you with your laboratory work. These are available online in convenient and compact pdf. Qiagen qiaprep mini prep dna purification method cn 27014 plasmid isolation procedure taken directly from the qiagen handbook with additional notation for clarity 1 resuspend the bacterial pellet in 250 l of buffer p1. Formulas for qiagen kit buffers for long term storage, all buffers should be sterilized by filtration or autoclaving.
Plasmid or cosmid dna purification using qiagen plasmid mega and giga kits. Techniques in molecular biology methods for plasmid dna isolation 3 other notes on this plasmid miniprep technique once cells have been lysed, mixing should be done thoroughly but gently, to avoid breaking plasmid and bacterial chromosomal dna. Solutions that contain ethanol, isopropanol or mops should be sterilized by filtration only. For uptodate licensing information and product specific disclaimers, see the respective qiagen kit handbook or user manual. Grow 25100 ml of bacteria culture for one midi prep and 100500 ml for maxi prep. Up to 10 mg highcopy plasmid dna is purified from 2. Provides protocol of isolating doublestranded dna from plasmid or cosmid by the alkaline lysis procedure followed by equilibrium ultracentrifugation in cesium chlorideethidium bromide gradients and. Emc skip over night culture go directly from plate into 100 mls of lb. Qiagen plasmid midi prep can be used to purify up to 100 mg of plasmid or cosmid dna. Use a high copynumber plasmid to obtain a good yield of plasmid dna. Add 24 ml 50 preps or 120 ml 250 preps 98100% ethanol to wn buffer before use. Compactprep plasmid megagiga purification handbook qiagen.
Background information, protocols, hints, and tips are provided for purification and analysis of plasmid dna, genomic dna, rna, and proteins, as well as recipes for buffers and solutions. Quick start procedure this quick start procedure is for. Genelute mammalian genomic dna miniprep 10 genelute mammalian genomic dna miniprep 70 genelute blood genomic dna kit 70 60506. Review and cite plasmid dna isolation protocol, troubleshooting and other methodology information contact experts in plasmid dna isolation to get answers. Alternatively, qiagentip 20 may be placed in qiarack 1 figure 1b. Invitrogen purelink quick plasmid miniprep kit was used in tacloning in order to qiagen plasmid mini prep kit was used to perform plasmid preparation. Protocol buffer c1 contains acetic acid, wear gloves or protective eyewear when handling. The plasmid preparation procedure is performed as usual. Purelink hipure plasmid dna megaprep and gigaprep kits. These are available online in convenient and compact pdf format at.
Qiafilterplasmidpurificationhandbook 042012 7 introduction qiafilter plasmid kits are based on the remarkable selectivity of patented qiagen resin. Qiagen endofree kit purelink expi kit qiagen protocol, troubleshooting and other methodology information contact experts in qiagen to get answers. Following is the miniprepqiagen kit protocol in biocoder, a highlevel programming language for expressing biology protocols. For qiagen plasmid midi, maxi, mega, and giga kit protocols. Zymopure ii plasmid kits are the fastest and simplest plasmid kits available to efficiently isolate transfectiongrade plasmid dna from e. Qiagen plasmid purification handbook sierra bermeja. Genomic dna prep protocol dasen lab genomic dna prep for pcr and southern 1. Qiaprep miniprep handbook washington state university. The qiafilter megagiga cartridge is designed for use with a 1 liter, 45mmneck glass bottle e. The new qiagen plasmid prep service provides ultrapure plasmid dna in milligram to. Start a 2 ml for mengo clones use starter culture in the morning from an isolated colony from an agar plate with the appropriate selective antibiotic. The qiaprep spin miniprep kit is designed for isolation of up to 20.
Preparation and lysis of bacterial cell cultures note. Here, we videorecorded the general procedures of miniprep through the qiagen s qiaprep 8 miniprep kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The range of protocols available is continually expanding, and additional qiagen protocols can. Qiaquick gel extraction kit protocol using a microcentrifuge. Qiagen plasmid mega and giga kits en print bookmark share pdf 299kb english. Qiagen plasmid mega and giga kits en print bookmark share pdf 299kb english format file size language download get adobe reader contact qiagen. The pureyield plasmid midiprep system is designed to isolate highquality plasmid dna for use in eukaryotic transfection and in vitro expression experiments. Qiagen plasmid purification handbook kirschner lab.
Centrifugation tubes or vessels with suitable capacity for the volumes specified in the appropriate protocol. These plasmid kits feature zymopure ii technology, including. Qiagen plasmid giga kit 5 from qiagen selectscience. Purifying plasmid dna from bacterial colonies using the qiagen miniprep kit. The qiacube is preinstalled with protocols for purification of plasmid dna, genomic dna, rna, viral nucleic acids, and proteins, plus dna and rna cleanup. I want to use a endofree giga prep kit from qiagene. Culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium. Add the provided rnase a solution to buffer mx1, mix and store at 4 c. Qiagentips may be placed in tubes using tip holders provided with the kits figure 1a. These are available online in convenient and compact pdf format. Qiagen plasmid purification handbook harvard university.
Setup of qiagentips awith tip holder or bwith qiarack 1. For purification of up to 10 mg transfectiongrade plasmid or cosmid dna. Cross reference of genelute to qiagen nucleic acid. Formulas for qiagen kit buffers genesee scientific. Qiagen tips can be left unattended, since the flow of buffer will stop when the meniscus reaches the upper frit in the column. All remaining components should be stored at room temperature. Rich media are not recommended for plasmid preparation with qiagentips. Purified dna usually is good enough for transfection and microinjection. More information about biocoder can be found on my home page.
Add 40 ml 50 preps or 180 ml 250 preps 98100% ethanol to buffer ws before use. After addition of ethanol, the sample is loaded onto an rneasy mini spin. Midimaxi plasmid prep column qiagen science gateway. Qiagen plasmid purification protocols are based on a modified alkaline lysis. Protocol qiaquick spin handbook 032001 23 qiaquick gel extraction kit protocol using a microcentrifuge this protocol is designed to extract and purify dna of 70 bp to 10 kb from standard or lowmelt agarose gels in tae or tbe buffer. They can be stored for at least 2 years without showing any reduction in performance, capacity, or quality of separation. Our technical service departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of qiagen prod ucts. What you see here is the autogenerated text ouput of the protocol that was coded up in biocoder see source code. Store solution irnase a at 28c after being combined see page 7. A protocol for isolation of plasmid dna for use in eukaryotic transfection and in vitro expression experiments. For the qiagen plasmid mini kit protocol microcentrifuge 1. Rneasy kits and rneasy protect kits are available in midi and maxi formats. The endofree maxiprep kit from qiagen or the genejet maxiprep kit from thermo fisher were used for maxi preparations. Instructions for use of products a2492, a2495, a2496.
For routine giga preparation of lowcopy plasmids, additional buffers p1. They are interchangeable and can be reused multiple times after treatment with hydrochloric acid, reequilibration, and wash. Unused columns can be cheaply purchased in bulk from epoch biolabs. The procedure can be performed in 40 minutes compactprep mega or 50 minutes compactprep giga and uses a vacuum pump and centrifuge. Should any product fail to perform satisfactorily due to any reason other. Do not vortex after cell resuspension, but mix by inversion. The qiagen plasmid giga kit uses gravityflow qiagen tip 0 anionexchange tips for efficient purification of plasmid dna. The purchaser must determine the suitability of the product for its particular use. Dna purification and analysis maximize sample yield, purity and integrity. This new plasmid dna isolation protocol will significantly reduce time and labor without increasing costs.
From these plasmid kits, the eluted plasmid dna is ready for immediate use, without any alcohol precipitation. Buffer c1 contains chaotropic salts, which may form reactive compounds when combines with bleach. The purelink hipure plasmid gigaprep kit also includes vacuum filtration columns that provide bacterial filtr. Endofree plasmid dna giga kit components are guaranteed for at least 12 months from the date of purchase when stored as follows. Plasmid or cosmid dna purification using qiagen plasmid mega and giga kits this protocol is designed for preparation of up to 2. Thomson instrument company is not affiliated with millipore, emd, qiagen, bio101 or their products step 1 step 2 miniprep determine dna concentration mgsl mega column. The purelink hipure expi plasmid gigaprep kit enables isolation of the highest levels of transfectiongrade plasmid dna from a single li giga prep using an enhanced anion exchange resin. The blue and purple qiagen columns are identical in formulation. Qiagen plasmid maxi prep can be used to purify up to 500 mg of plasmid or cosmid dna. Very likely this protocol can be used with other similar columns. Typical yields from low copy number plasmids are highly dependent upon culture conditions and vectorhost strain combinations.
If you have questions about product specifications or performance, please call qiagen technical services or your local distributor see back cover or visit. Purifying plasmid dna using the qiagen miniprep kit protocol. Up to 400 mg agarose can be processed per spin column. No cell clumps should be visible after resuspension of the pellet. The ultrafast purification protocol, based on the remarkable selectivity of patented qiagen resin, allows the isolation of ultrapure, supercoiled plasmid dna. The purelink hipure plasmid gigaprep kit also includes vacuum filtration columns that provide bacterial filtration without centrifugation, enabling.
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