The Marine Animal Tissue Genomic DNA Purification Kit provides a simple and rapid method for high quality genomic DNA purification from tissues(either fresh or frozen at -70°C until use) of marine animal, such as fish, shrimp, shellfish or crab. The Genomic DNA system uses the silica-gel-membrane technology for simple and fast isolation of Genomic DNA without phenol/chloroform. Homogenization is not necessary since tissues are directly lysed by Proteinase K. The buffer system is optimized to allow selective binding of DNA to the silica-gel membrane. The simple centrif µgation protocol completely removes contaminants such as proteins, divalent cations, and secondary metabolites. Pure DNA is then eluted in water or low-salt buffer, ready to use. The typical yield of genomic DNA is 3-35 µg from 10 mg of tissue. The purified high molec µLar weight genomic DNA is suitable for direct use in all common molec µLar biology applications : PCR, restriction digestion, cloning, DNA sequencing and Southern blot analysis.
Features
Efficient : 3-35 µg of genomic DNA from 10 mg of tissue or 1 x 106-107 c µLtrue cells.
Fast : Procedure takes only 30 min.
Universal : Purifies genomic DNA from various sources.
Safe : No phenol/cholroform extraction step.
High Purity : Purified DNA is ready for downstream application such as PCR, restriction digestion.
Downstream Applications
Purified DNA is free from contaminants and enzyme inhibitors, and typically has A260/A280 ratios between 1.7 and 1.9, and is suitable for applications such as :
ü Restriction digestion
ü PCR
ü Labeling
ü Library construction
Kit Content : (for a 50-prep kit Volume)
Component
Volume or Volume
Solution DS
15 ml
Solution MS
20 ml
Proteinase K
1 ml
Wash Buffer (PS)
30 ml
Wash Buffer (PE)
15 ml
Elution Buffer TE
5 ml
Spin Columns
50 each
Storage
Store Protein K at -20℃, other reagents can be stored at room temperature for up to 1 year. Any precipitate in the Solution DS and Solution MS can be re-dissolved by incubating at 37°C before use.
Important Notes
• Prior to the initial use of the kit, dilute the Wash Buffer(PE) with ethanol (96-100%) :
N1172(100preps)
Wash Buffer(PE)
30 ml x2
Ethanol
, , 90 ml x2
Total Volume
120 ml x2
After the ethanol has been added, mark the check box on the bottle to indicate the completed step.
• Examine the solution for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37°C and cooling to 25°C.
• All purification steps sho µLd be carried out at room temperature.
Protocol
1.Marine animal tissue (either fresh or frozen at -70°C until use) can be processed by freezing with liquid nitrogen and grinding into a fine powder using a mortar and pestle. Add 10mg of this tissue powder to a 1.5ml microcentrif µge tube. Do not exceed 10 mg or it will reduce the lyse efficiency.
2.Add 200 μl Solution DS. Vortex vigorously to resuspend cells.It is essential that the sample and Solution DS are mixed immediately and thoro µghly by vortexing or pipetting to yield a homogeneous solution.
Optional If RNA-free genomic DNA is required, add 4 μl RNase A (100 mg/ml) and incubate for 5 minutes at room temperature. RNase A (100 mg/ml) can be purchased separately .
3.Add 20 μl Proteinase K. Mix thoro µghly by vortexing. Incubate at 55℃ and inverting at times till a clear homogeneou solution is produced, spin down the water beads from the wall of the tube.
4.Add 220 μl Solution MS, Mix thoro µghly by vortexing. Incubate at 65°C for 10 minutes to yield a homogeneous solution. Spin down the water beads from the wall of the tube.
5.Add 220 μl ethanol (96–100%) to the sample, and mix thoro µghly by inverting. A precipitate may appear. Pipet the mixture from step 4 into the spin column placed in a 2 ml collection tube (provided). Centrif µge at 12,000rpm for 1 min. Discard flow-thro µgh.
6.Add 500 μl Wash Buffer PS, and centrif µge for 1 min at 12,000 rpm. Discard flow-thro µgh.
7.Add 500 μl Wash Buffer PE, and centrif µge for 1 min at 12,000 rpm. Discard flow-thro µgh. Repeat step 6 again.
8.Centrif µge for 3 min at 12,000 rpm to dry the column membrane. Discard flow-thro µgh and collection tube.
Note It is important to dry the membrane of the spin column, since residual ethanol may interfere with subsequent reactions. This centrif µgation step ensures that no residual ethanol will be carried over during the following elution.Following the centrif µgation step, remove the spin column caref µLly so that the column does not come into contact with the flow-thro µgh, since this will res µLt in carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrif µgation for 1 min at 12,000 rpm.
9.Place the spin column in a clean 1.5 ml microcentrif µge tube (not provided), and pipet 30-100 μl Eluent Buffer AE (prewarm to 65℃)directly onto the membrane. Incubate at room temperature for 2 minutes, and then centrif µge for 2 min at 12,000 rpm to elute. The tube contains the purified DNA. Store the DNA at -20℃.