Plasmid dna purification protocol featuring the pureyield plasmid midiprep system 14 a. Inoculate 5ml lb containing the appropriate antibiotic with a single colony. Streak out strain of interest onto lb plate with the appropriate antibiotic selection. Qiagen plasmid purificationhandbook 042012 6 data sheets msdss. Qiaprep spin miniprep kit protocol using a microcentrifuge this protocol is designed for purification of up to 20 g of highcopy plasmid dna from 15ml overnight cultures of liin lb luriabertani medium.
The plasmid preparation procedure is performed as usual. Qiagen protocols are optimized for use with yeast cultures grown in standard ypd media see page 57 to a cell density of approximately 3 x 10 8 cellsml. These are available online in convenient and compact pdf format. The range of protocols available is continually expanding, and additional qiagen protocols can. Special applications 44 purification of lowcopy plasmids and cosmids 44 purification of plasmid dna prepared by other methods 44 references 45 ordering information 46 qiagen distributors 51. Rich media are not recommended for plasmid preparation with qiagen tips. Qiagen plasmid purification handbook sierra bermeja.
Nov 29, 2016 qiagen allprep dnarna ffpe kit protocol pugh lab printed. The system provides a rapid method for purification using a silicamembrane column. Throughout the remainder of this document the supplied cell resuspension solution cra, cell lysis solution cla and neutralization solution nsb are referred to. Sample preparation using 200mg of starting material this protocol is intended to provide directions for isolating genomic dna from up to 200mg dry weight of food material per isolation. Agarose gel analysis of plasmid dna 45 references 45. Key steps in the qiagen largeconstruct protocol 25 preparation of the cell lysate 25 exonuclease digestion of chromosomal dna 26 dna binding and washing on the qiagen tip 26 desalting and concentration 26 appendix a 27 protocol for high yields of largeconstruct dna without removal of genomic dna 27 appendix b 31 composition of buffers 31. I believe the promega kit does not remove endotoxins. On rna with rneasy micro kit is the kit delivers highly reproducible ct values were from small amounts of genomic and rna. Qgen quick quote qgen free report recently announced the launch of its qiaseq direct sarscov2 kit. The system incorporates a unique endotoxin removal wash to remove protein, rna and endotoxin contaminants from purified plasmid dna. 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. Dna purification by centrifugation prepare lysate 1. Qiaquick pcr purification kit protocol using a microcentrifuge this protocol is designed to purify single or doublestranded dna fragments from pcr and other enzymatic reactions see page 8. The procedure can be performed in 20 midi and maxi, 40 mega, or 50 minutes giga using a vacuum pump and centrifuge.
For cleanup of other enzymatic reactions, follow the protocol as described for pcr samples or use the minelute reaction cleanup kit. The dilution factor should then be used in calculating the number of cells per ml. We therefore encourage you to contact us if you have. Highthroughput genomic dna isolation systems for blood 19. A protocol for isolation of plasmid dna for use in eukaryotic transfection and cellfree expression experiments. The qiagen tip10,000 giga column is intended to deliver up to 10 mg of high copynumber plasmid dna from escherichia coli. Lysis efficiency can be improved by cell disruption using a rotorstator homogenizer, such as the qiagen tissueruptor, or a bead mill, such as the qiagen tissuelyser. For purification of lowcopy plasmids and cosmids, large plasmids 10. We have found that by repeatedly regenerating the column and reapplying the flowthrough volume of dna extract, we can readily obtain 24fold increased yields, up to 40 mg, from the standard 2. This information is helpful to other scientists as well as to the researchers at qiagen. Qiagen plasmid purification handbook 082003 7 endofree plasmid kits endofree plasmid kit maxi 10 mega 5 giga 5 catalog no.
Preparation and lysis of bacterial cell cultures note. Qiagen sample and assay technologies qiagen is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Qiafilter mega giga cartridges are special filter units which operate with any vacuum source to clear bacterial lysates from up to 2. Qiagen customers are a major source of information regarding advanced or specialized uses of our products. Sample preparation using the qiacube follows the same steps as the manual procedure i. A supplementary protocol allowing the simultaneous disruption of up to 48 tissue samples using the qiagen tissuelyser is available from qiagen technical services. Qiagen s dna lood mini kit or equivalent, a process that takes about 1 hour to complete. What is your preferred maxi prep kit for plasmid dna grown. Kit contents qiagen onestep rtpcr kit 25 100 catalog no. The purified dna is equivalent to that obtained by 2 x cscl gradient centrifugation and is suitable for transfectiongrade applications. These are available online in convenient and compact pdf format at.
This week i tried the pro mega wizard maxi kit, the invitrogen pure link with filter, and qiagen plasmid plus. Compactprep plasmid megagiga purification handbook qiagen. Wizardr magnetic dna purification system for food technical. Highyield protocols and extra buffer volumes are provided with the qiagen plasmid plus kits. Qiagen plasmid purificationhandbook 042012 9 important notes please take a few moments to read this handbook carefully before beginning the dna preparation. The protocols differ primarily in the lysis and homogenization of the sample. The bionano prep blood dna isolation protocol targets recovery of white blood cells wbc using differential red blood cell rbc lysis. Rich media are not recommended for plasmid preparation with qiagentips. Also be sure to read and follow the appropriate detailed protocol. The kit is a viral genome enrichment and library preparation solution that.
Qiaamp dna mini kit and qiaamp dna blood mini kit handbook. Fifty microliters of magnesil pmps step 8 allows the purification of up to 5g of dna eluted in 100l of water step 14. These are available online in convenient and compact pdf. Qiagen tips are ideally suited for rapid and simple preparation of 112 samples. Prep size overnight bacterial culture volume approximate yield miniprep 15 ml up to 40 g midiprep 25100 ml up to 200 g maxiprep 100500 ml 750850 g megaprep 500 ml2. For the qiagen plasmid mini kit protocol microcentrifuge 1. The new qiagen plasmid prep service provides ultrapure plasmid dna in milligram to.
Qiagen plasmid plus mega and giga kits en print bookmark share pdf 57kb english format file size language download get adobe reader contact qiagen. If qiagen plasmid purification kits are new to you, please visit our. If qiagen plasmidpurification kits are new to you, please visit our plasmid resource page at and click on the link general considerations for optimal results. In this handbook, different protocols are provided for different starting materials. Boundary court gatwick road crawley west sussex, rh10 9ax orders 01293422911 fax 01293422922 technical 01293422999 usa qiagen inc. Throughout the remainder of this document, the supplied cell resuspension solution cra, cell lysis. Qiagen plasmid purification protocols are based on a modified alkaline lysis. For purification of lowcopy plasmids and cosmids, large plasmids 10 kb, and dna prepared using other. Tested with rneasy micro kit qiagen protocol, in the developmental stage, and northern blot of high quality and growth conditions.
These are available online in convenient and compact pdf format at where you can find, view, and print the msds for each qiagen kit and kit component. Repeated use of qiagen columns in largescale preparation. Qiagen plasmid purification handbook harvard university. Protocols qiaprep spin miniprep kit using a microcentrifuge 23 using 5 ml collection tubes 24. Qiagen plasmid plus mega and giga kits en print bookmark share pdf 57kb english format. Principle and procedure qiagen plasmid purification protocols are based on a modified alkaline lysis.
Plasmid dna prep large scale cscl2 prep growth and amplification. Plasmid dna purification products, prep sizes and approximate yields. A rapid protocol to isolate highquality plasmid dna, using a silica membrane column, in less than 10 minutes, depending on the number of samples. Qiagen provides a variety of qiaamp genomic dna kits for fast. Standard dna purification protocol this section details our standard protocol for lysis of bacterial cell cultures and dna purification. For higher yields and purity use the alternative protocol below to harvest and process up to 3ml of bacterial culture. The resulting wbcs are quantitated, immobilized in agarose plugs, and subjected to plug. Qiagen tips can be left unattended, since the flow of buffer will stop when the meniscus reaches the upper frit in the column. Principle and procedure qiagen plasmid purification protocols are based on a modified alkaline lysis procedure, followed by binding of plasmid dna to qiagen anionexchange resin.
Qiagen continually strives to streamline nucleic acid purification. Plasmid dna purification using compactprep plasmid mega kits. Centrifugation tubes or vessels with suitable capacity for the volumes specified in the appropriate protocol. Each disposable qiagen tip is designed to operate by gravity flow, reducing the amount of handson time required for the purification procedure. Qiagenplasmidpurificationhandbook 042012 5 qiagen mega 5 mega 25 giga5 plasmid plasmidkit bufferset catalogno. Our advanced, highquality products and services ensure success from sample to result. After drain disposal, please flush with at least 1020 fold excess of water to thoroughly rinse out the sink and sink trap, and to dilute the. Once the sample is applied to the allprep dna spin column, the protocols are similar see flowchart, next page. Repeated use of qiagen columns in largescale preparation of. For parallel processing of more than one compactprep mega preparation or for compactprep giga preparations on the qiavac 24 plus.
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