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| DNA fragments for probe DNA or ligation must be separated and purified from other DNA fragments. MEGA-spinTM employs a column method to purify target DNA in excised agarose gel. The column method uses a highly concentrated salt solution to keep the target DNA bound to the column membrane. The binding reaction occurs due to the disruption of the organized structure of water molecules and the interaction with the nucleic acids. Thus the adsorption to the specifically pretreated membrane is favored. Since the binding process is specific for nucleic acids, the bound material can be separated and purified from impurities e.g. salts and proteins, with simple washing step. Nucleic acids elute from the column membrane in a low salt buffer or water.
MEGA-spinTM is designed to extract and purify DNA of 100bp to 10kb from standard or low-melt agarose gels in TAE or TBE buffer. Furthermore, the kit guarantees a high yield of purification up to 70-90%. DNA fragments isolated with MEGA-spinTM Agarose Gel Extraction Kit are efficiently ligated into plasmid cloning vectors or specifically labeled using either random primed labeling or nick translation. No inhibition of digestion with restriction endonucleases is observed.
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- Extract and purify DNA of 100bp to 10kb from all types of agarose gel
- Recovery between 70 and 90%
- Spin column technology to recover DNA from excised agarose bands
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| - Sequencing, cloning, ligation, probe labeling
ligation, random primed labeling, nick translation and etc.
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| * Agarose
Lysis Buffer |
180㎖ |
* Washing
Buffer
: Add Absolute EtOH 200ml before use. |
50㎖ |
| * Elution
Buffer |
20㎖ |
* Columns
: Column contains silica membrane |
250
columns |
* Collection
Tubes
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250
Tubes |
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 Purification
yield |
| 1. % Recovery of DNA from the different fragment length
& amount of DNA |
| [ Table. the different fragment length ] |
[ Table. the amount of DNA ] |
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Fragment
length |
%
Recovery |
|
100bp - 200bp |
80% |
| 200bp
- 4Kb |
90% |
| 4Kb
- 10Kb |
85% |
| >
10 Kb |
60% |
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Amount
of DNA |
%
Recovery |
| 5 - 10ug |
90% |
| 10 -
30ug |
85% |
| > 30ug |
70% |
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| 2. Recovery of DNA from different PCR product size |
Purification of DNA fragments with the MEGA-spinTM Agarose
Gel Extraction Kit is based on solubilization of agarose and selective
adsorption of nucleic acids onto MEGA-spinTM silica-membrane
in the presence of charotropic salt. The different sizes of DNA fragments
can be extracted from gels in 70%-90% recovery.
Fig. DNA fragments extraction with the MEGA-spinTM
Agarose Gel DNA Extraction Kit
DNA fragments were extracted from gels and pooled after extraction.
Recoveries of approximately more than 70%-90% are obtained for different
sizes (100bp-10kb).
Lane M1, M2, Marker DNA;
Lane 1, 6, 131bp DNA; Lane 2, 7, 570bp DNA;
Lane 3, 8, 1.3Kb DNA; Lane 4, 9, 4.5Kb DNA;
Lane 5,10, 10Kb DNA |
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| Purification
purity |
1.
Enzyme digestion result |
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Fig.
Enzyme digestion
3kb PCR product was extracted using MEGA-spinTM
Agarose Gel Extraction Kit and then digested with EcoRI.
The digestions were Performed at 37¡E for
1hr with each enzymes. And then result was analyzedin
gel electrophoresis.
Lane M, Marker
DNA; Lane 1, 3, undigested PCR product,
extracted using MEGA-spinTM Kit; Lane
2,4, digested PCR product |
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| Comparison
of DNA yield from different company kit |
The MEGA-spinTM
Kit was compared with other supplier's products in the recovery
of DNA fragments from gels. Comparison of various products
at same conditions showed that the recovery of iNtRON's MEGA-spinTM
Kit was similar or excellent to other products. |
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Fig.
Comparison of different supplier's products
The DNA fragments (4.5Kb and 1.3Kb) were extracted from gels
using kits from different suppliers.
1.3Kb DNA fragment; 4.5 Kb DNA fragment
Lane M, Marker DNA; Lane 1,
MEGA-spinTM Kit; Lane 2, Supplier
A; Lane 3, Supplier B;
Lane 4, Supplier C; Lane 5, Supplier
D |
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