PrestoSpin Duniversal Kit

Mini Spin Kit

Extraction of genomic and plasmid DNA

With  Protocols for fast purification of PCR-ready DNA

Arguments

July 2002

Introduction

At first it is important to know from which kind of biological material the customer extracts DNA. The following main groups may be distinguished

1. Plasmid DNA

   1. Mini

   2. Midi

   3. Maxi

2. Genomic DNA

   1. Phages (Lambda)

   2. Viruses

   3. Bacteria

   4. Yeasts

   5. Fungi

   6. Plants

   7. Animal and Human Tissue

   8. Blood

   9. Cell Culture

   10. Soils and Sediments

   11. Food and Feed

3. DNA Purification

   1. Gel Extraction

   2. PCR Purification

   3. Nucleotide Removal

1. Mostly, for each application a special kit is used. Disadvantages are:

   1. The diverse kits are piled up in the laboratory

   2. You have to work with many different handbooks and procedures

   3. Although you only want to do a few special extractions you have to buy a complete kit.

   4. Many kits reach their expiry date, although they were not entirely used

2. Molzym offers a kit for all DNA extractions from any biological material, i.e.

   1. You have to buy only one kit for all DNA extractions

   2. You need only one detailed and colored handbook with simple protocols

   3. The DNA purification procedure is always the same

   4. Probably, the kit will never reach its expiry date because it will be completely used before

3. All suppliers of DNA extraction kits use the same technology: the matrix material is silica.

4. Molzym´s kit is based on a new patent-pending technology called “cation bridging”

   1. The matrix material is clay

   2. In presence of multivalent cations the DNA polyanion binds to the negatively charged clay (“cation bridging”)

   3. A complexing agent eliminates the cations

   4. Pure DNA is eluted with TE buffer

5. Whether Mini or Midi purification – each time a special kit is used.

6. Molzym columns yield up to 80 µg genomic or 50 µg plasmid DNA. Additionally, fast protocols for PCR applications are included.

7. Midi kits of other suppliers are extremely expensive.

8. With the GLuniversal DNA Minispin Kit, each extraction has the same price – it does not make any difference whether you extract 1 µg DNA from plant material for PCR or 80 µg DNA from bacteria for hybridization.

9. Many kits of other suppliers are long-during, difficult to understand and do result in impure DNA.

10. Not with the GLuniversal DNA Minispin Kit:

    1. The A_260/280 quotient lies always between 1.7 and 2.0

    2. You need only 10 to 25 min from cell lysate to pure DNA

11. Silica-based systems tend to become blocked by proteins, polysaccharides and other high molecular weight substances.

12. Molzym´s columns are not blocked. Indeed,  proteinase K is not necessary and even DNA from slimy snails can be extracted without any problems.

13. In silica-based systems, mostly an RNase digestion is necessary during DNA extraction since RNA und DNA have the same binding sites.

14. This is not due to the Molzym Matrix. RNase digestion is optional but not necessary. If small amounts of RNA are tolerated you do not need an RNase digestion, e.g. for applications like cloning, PCR or transformation. For other applications like sequencing or transfection an RNase digestion is performed on the column.

FAQ

Which components are included in the GLuniversal DNA Minispin Kit?

All solutions, buffers, RNase and Proteinase necessary for the extraction of plasmids, genomic DNA from phages , prokaryotes, yeasts, fungi, plants, animals, blood, cell culture and soils are included, except:

• 70% Ethanol for washing steps

• Isopropanol for plasmid preparation

• lyticase for yeast lysis (can be ordered separately from us)

• lysozyme for bacterial lysis (can be ordered separately from us)

• DNase/RNase for nucleic acid digestion in phage suspensions before extraction (can be ordered separately from us)

• Extraction Buffer for soil

What is the size of genomic DNA that is obtained with GLuniversal Minispin Columns?

The molecular weight of genomic DNA strongly depends on the lysis procedure of the biological material. Mechanical lysis procedures cause shearing of DNA which leads to molecular weights of 20-40 kb. If high molecular weight DNA >40 kb is wanted, enzymatic lysis methods have to be preferred.

Shearing of plasmids leads to single strand breaks and to open circular plasmid DNA. With the GLuniversal DNA Minispin Kit almost all plasmids are covalently closed circular („normal“ plasmids, i.e. <5 kb)

Why is the O.D. ratio of DNA purified using GLuniversal DNA Minispin Kit high?

A₂₆₀/ A₂₈₀ ratio for purified nucleic acids is high. High level of residual RNA is due to unsufficient RNA digestion. Check culture volume against recommended volumes, and reduce if necessary. If RNase is more than 6 months old or was not properly stored, add new RNase A.

Why did I get low plasmid yield? The importance of an analytical gel?

Poor yields and quality can be caused by a number of different factors. To determine at what stage of the procedure any problem occurred, save fractions from different steps of the purification procedure, and analyze by agarose gel electrophoresis.

Why is my plasmid DNA contaminated or poor quality?

Genomic DNA in the eluate.

Mixing of bacterial lysate was too vigorous. The lysate must be handled gently after addition of Buffers AL and NB to prevent shearing of chromosomal DNA. Reduce culture volume if lysate is too viscous for gentle mixing.

RNA in the eluate.

RNase A digestion was insufficient. Check culture volume against recommended volumes, and reduce if necessary. If RNase is more than 6 months old or was not properly stored, add new RNase A.

Nuclease contamination.

Check buffers for nuclease contamination and replace if necessary. Use new glass-and plasticware, and wear gloves.

Lysis time was too long.

Ensure that lysis step does not exceed 5 min.

Overloaded alkaline lysis.

Check the culture volume and yield against the capacity of the column. Reduce the culture volume accordingly or alternatively increase the volumes of Buffers RS, AL, and NB.

Shearing during redissolving.

Redissolve DNA gently, without vortexing or vigorous pipetting. Avoid using small pipet tips.

What are the plasmid copy numbers of various plasmids?

Plasmids vary widely in their copy number (refer to the table below), depending on the origin of replication they contain (pMB1 or pSC101 for example) which determines whether they are under relaxed or stringent control; as well as the size of the plasmid and its associated insert. Some plasmids, such as the pUC series and derivatives, have mutations which allow them to reach very high copy numbers within the bacterial cell. Plasmids like pNOF100 or plasmids based on pBR322 and many cosmids are generally maintained at lower copy numbers. Very large plasmids are often maintained at very low copy numbers per cell.

Please note: the copy number of plasmids and cosmids can be substantially influenced by the cloned insert. For example, a high-copy pUC plasmid may behave like a medium-or low-copy plasmid when containing certain inserts (e.g. very large DNA fragments), resulting in lower DNA yields than expected.

Origins of replication and copy numbers of various plasmids and cosmids¹

Comparison of the Molzym Minispin Kit with Qiagen Kits:

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