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Protease K digestive lysing method is suitable for the digestion and treatment of all specimens, especially
DNA
samples. Such as tissue woven cells (including paraffin encased tissue) fluff, hair, fine spots, blood (
serum
, plasma, whole blood) local secretions, urine, feces and so on.. 1.
reagents
(1) protease K digestives: 10mmol/L Tris.cl (PH8.0)
10mmol/L
EDTA
150mmol/LNaCL
0.5%
SDS
100 to 200ug/ml enzyme protein.
(2) protease K preferably with water into 20 mg/ml, ready to use to add digestive fluids.. 2. Extraction methodSome specimens, before digesting with protease K, also need to be pre-treated, such as feces, secretions, sputum, tissue, paraffin encased tissue, etc. , the method has centrifugal remove impurities, dewax and so on.
clinical specimens or pre-treated specimens plus protease K lysate 50 to 100ul. Mix well, 55 degrees C 1 to 3 hours, or 37 degrees C overnight. Add the saturated phenol of the same volume 1 to 2 times, and then add the same volume of chloroform: isoquinol (49:1) pumping once, add 1/10 volume of pH 5.23mmol /L sodium acetate buffer.
add 2.5 times the volume of ice-cold waterless ethanol (or equal volume of isopropyl alcohol) - 20 degrees C placed at least 3h. After removal 14000 rpm centrifugation 15min. Careful absorption or pour out of the upper clear, precipitation add 75% ice-cold ethanol 15000r/min5min departure Heart washing 1 to 2 times, especially careful to absorb or pour out the upper clear, vacuum or 37 degrees C temperature box or room temperature
drying
, plus TE buffer 20ul. Dissolved, take 3 to 8ul for
PCR
amplification, or put -20 degrees C to save.
Protease K digestion method in addition to the above-mentioned classical treatment method, can also be in protein K digestion and treatment specimens, after centrifugal treatment, absorption of 95 to 97 degrees C or boiling 10min inactivated protease K, direct nucleic acid template for PCR amplification. If there are more impurities, can also be used for PCR reaction after phenol: chloroform pumping. This method
protein
other impurities eliminated completely, Taq enzyme activity is not affected, with good repeatability and stability. But the operation is complicated and the technical requirements are high..