
BP-10 Spin Column Genomic DNA Isolation Kit (For Animal)
Kit Contents:
Components | GDMIP-A50 | GDMIP-A100 |
ACL Solution PBS Solution AB Solution Proteinase K Wash Solution Elution Buffer BP-10 Column 2.0-ml Collection Tube | 20 ml 20 ml 20 ml 20mg 12 ml 5 ml 50 50 | 40 ml 40 ml 40 ml 40mg 24ml 10 ml 100 100 |
A.Before use, add 1ml or 2ml of sterilized water to the tube containing 20mg or 40mg of proteinase K, keep at -20 oC.
B.Before use, add 48ml of 100% ethanol to 12ml Wash Solution for GDMIP-50, add 96ml of 100% ethanol to 24ml Wash Solution for GDMIP-100. For other volumes of wash solution, simply add enough ethanol to make a 4:1 ratio (volume of added ethanol : volume of Wash Solution = 4:1).
C.Elution Buffer is 2.0 mM Tris-HCl pH 8.0-8.5. Although TE buffer pH 8.0 or water can be used, yield is generally 10% lower.
Storage:
With the exception of the Proteinase K, the kit may be stored at room temperature. The proteinase K should be stored at 4ºC. The kit is stable for 12 months at room temperature. For longer storage, keep all contents cold.
Principle:
This kit is designed for fast isolation of genomic DNA from animal tissues. The kit contains a membrane embedded in BP-10 spin column for binding up to 10ug of genomic DNA. Nucleotides, proteins, salts, and other impurities do not bind to the column. Purified genomic DNA can be applied in most molecular biology experiments including restriction digestion, PCR, Southern-blotting etc.
Applications:
Genomic DNA purification from different animal tissues.
Features:
- Preparation of high quality genomic DNA from variable sources.
- Rapid and economical.
- High yields
- No phenol / chloroform extraction , no ethanol precipitation
Procedure for Isolation of Genomic DNA from Variable Sources.
For Animal Tissue
- Cut up to 30mg tissue and place in a 1.5ml centrifuge tube.
- Add 300ul of ACL Solution (Animal Cell Lysis Solution) to 1.5ml centrifuge tubes and 20ul proteinase K.
- Incubate at 55oC until the tissue is completely lysed (usually 1-3 hours). Occasionally vortex. Incubation in shaking water bath can reduce lysis time.
- Cool to room temperature. Vortex for 20 seconds and Centrifuge 12000 rpm for 5 minutes.
- Pipette 300ul of supernatant into an BP-10 spin column (if pellet not visible repeat previous step) and add 300ul of AB Solution . Mix by occasionally inverting tube and keep for 2 minutes.
- Centrifuge 4000 rpm for 2 minutes and discard the flow-through.
- Add 500ul of Wash Solution, and spin at 8,000 rpm for 1 minute.
- Repeat washing step 7.
- Discard flow-through. Spin at 10,000 rpm for an additional minute to remove residual amount of Wash Solution.
- Place the column into a clean 1.5 ml centrifuge tube. Add 30-50ul Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50oC for 2 minutes. Incubate at 37 or 50oC could increase recovery yield.
- Spin at 10,000 rpm for 1 minute to elute DNA from the column.
- Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb.
For Rodent Tail
- Place numbered 1.5ml centrifuge tubes in dry ice.
- Cut 0.5cm to 1cm from end of tails and place in tubes.
- Add 300ul of ACL Solution to 1.5ml centrifuge tubes and 20ul proteinase K.
- Incubate at 55oC overnight with rocking or for several hours with occasional mild vortexing every 15 minutes.
- Cool to room temperature. Vortex 20 seconds and Centrifuge 12000 rpm for 5 minutes.
- Pipette 300ul of supernatant into an BP-10 spin column (if pellet not visible , repeat previous step) and add 300ul AB Solution . Mix by occasionally inverting tube and keep for 2 minutes.
- Centrifuge 4000 rpm for 2 minutes and discard the flow-through.
- Add 500ul of Wash Solution, and spin at 8,000 rpm for 1 minute.
- Repeat washing step 8
- Discard flow-through. Spin at 10,000 rpm for an additional minute to remove residual amount of Wash Solution.
- Place the column into a clean 1.5ml centrifuge tube. Add 30-50 ul Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50oC for 2 minutes. Incubate at 37 or 50oC could increase recovery yield.
- Spin at 10,000 rpm for 1 minute to elute DNA from the column.
- Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb.
For Cultured Animal Cell
- Centrifuge the appropriate number of cells(>5x106) for 5 minutes at 1200 rpm.
- Resuspend pellet in 200ul of PBS Solution.
- Add 20ul of proteinase K .
- Incubate at 70oC for 10 minutes.
- Cool to room temperature. Vortex for 20 seconds and Centrifuge 12000 rpm for 5 minutes.
- Pipette 200ul of supernatant into an BP-10 spin column (if pellet not visible , repeat previous step) and add 200ul AB Solution. Mix by occasionally inverting tube and keep for 2 minutes.
- Centrifuge 4000 rpm for 2 minutes and discard the flow-through.
- Add 500ul of Wash Solution, and spin at 8,000 rpm for 1 minute.
- Repeat washing step 8
- Discard flow-through. Spin at 10,000 rpm for an additional minute to remove residual amount of Wash Solution.
- Place the column into a clean 1.5ml centrifuge tube. Add 30-50ul Elution Buffer into the center part of membrane in the column. Incubate the tube at RT for 2 minutes. To incubate at 37 or 50oC could increase recovery yield.
- Spin at 10,000 rpm for 1 minute to elute DNA from the column.
- Measure DNA quantity by UV absorption at A260(1.0 OD unit is equivalent of 50ug). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb.
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大多数药物在体内都是和特异性受体相互作用,改变细胞的生理生化功能而产生效应。目前已经确定的受体有30多种,根据受体存在的标准,受体可大致分为三类:
1.细胞膜受体:位于靶细胞膜上,如胆碱受体、肾上腺素受体、多巴胺受体、阿片受体等。
2.胞浆受体:位于靶细胞的胞浆内,如肾上腺皮质激素受体、性激素受体。
3.胞核受体:位于靶细胞的细胞核内,如甲状腺素受体。
另外也可根据受体的蛋白结构、信息转导过程、效应性质、受体位置等特点将受体分为四类:
1.含离子通道的受体(离子带受体):如N-型乙酰胆碱受体含钠离子通道。
2.G蛋白偶联受体:M-乙酰胆碱受体、肾上腺素受体等。
3.具有酪氨酸激酶活性的受体:如胰岛素受体。
4.调节基因表达的受体(核受体):如甾体激素受体、甲状腺激素受体等。
有些受体具有亚型,各种受体都有特定的分布部位核特定的功能,有些细胞也有多种受体。
请教各位大神,我最近在做WB,565KD的蛋白,动物组织的样品,听说样品制备需要蔗糖裂解液,请问这个是必须的嘛?用一般蛋白提取方法制备可以吗?还有电泳和转膜条件能否分享一下。不胜感激

