Manual - Omega Bio-Tek
Contents
1. Contents PMPOCMIG HONS a isea ale od aiia eRe Tastee be R tes ee QR Masses Einar eh od Principle cder ee einen abet Gabe dO Sere ee Storage and Stability n a naaa et aks Set ehea ee eae eet PCI Contents eate ea pas eases Mane ce ande Salar E a aO E A amp ant 10 25 mg SQ Plant DNA Protocol aaua anaana aaa 100 200 mg SQ Plant DNA Protocol nnana aaa aaa 500 mg SQ Plant DNA Protocol aaau auaa aaaea Troubleshooting Guide a na naaa eee eee Revised January 2008 2 2 Introduction SQ Plant DNA Kit is designed for rapid and reliable isolation of total DNA from various plant samples The SQ Plant DNA Kit uses a proprietary buffer system to remove polysaccharides and proteins to isolate high molecular weight genomic DNA There is no toxic substance such as phenol chloroform or guandine salts involved in this system The system can be easily scaled up or down allowing for the purification from various amounts of starting materials Principle If using the SQ Plant DNA Kit for the first time please read this booklet to become familiar with the procedure and its various modifications Samples are first lysed in a specially formulated buffer The protein is precipitated by adding SQ2 After removal of the protein the supernatant is mixed with 1 volume of isopropanol to precipitate the DNA The DNA pellet is washed with 70 ethanol and dissolved with water or low ionic strength buffer Purified DNA can be directly us2. ed in downstream applications without the need for further purification Storage and Stability All components of the SQ Plant DNA Kit are stable for at least 12 months from date of purchase when stored at Room Temperature except Rnase A which should be stored at 2 8 C During shipment or storage in cool ambient conditions precipitates may form in the some of the buffers Dissolve such deposits by warming the solution at 37 C Kit Contents SQ Plant DNA Kit SQ Plant DNA Kit Product No D3095 00 D3095 01 D3095 02 Amount of Tissue processed per 1 Gram 10 Grams 40 Grams kit SQ1 35 ml 350 ml 2x700 ml SQ2 12 ml 120 ml 500 ml RNase A 110 ul 1 1 ml 4x1 1 ml EB Buffer 3 ml 30 ml 120 ml User Manual 1 1 1 Buffer EB 10 mM Tris Hcl pH 8 5 A 10 25 mg SQ Plant DNA protocol Material and Equipments supplied by User Have the following reagents and supplies ready before starting procedure Table top centrifuge capable at least 13 000 xg 1 5 or 2 ml Nuclease Free centrifuge tubes 70 ethanol 100 Isopropanol waterbath or incubator Before starting m Preheat a water bath or incubator to 65 C m Warm up the SQ1 at 37 C water bath 7 Under cool ambient conditions crystals may form in some of the buffers This is normal and the bottle should be warmed to re dissolve the salt Procedure 1 Add 750 uL of SQ 1 Buffer to a 1 5 mL Centrifuge Tube Grind fresh plant tissue finely in liquid ni
3. imes Centrifuge at 4 000 x g at room temperature for 15 minutes Carefully remove the supernatant and drain tube on a clean absorbent paper Air dry the DNA pellet for 10 minutes Add 500 ul of Buffer EB to the tube Vortex the tube for 10 seconds Allow the DNA to re hydrate at 65 C for at least 30 minutes Store the sample at 80 C C 500 mg SQ Plant DNA protocol Material and Equipments supplied by User Have the following reagents and supplies ready before starting procedure m Centrifuge capable at least 4 000 x g 7 70 ethanol m 100 Isopropanol 7 50 mL Centrifuge TubeS m waterbath or incubator Before starting 7 Preheat a water bath or incubator to 65 C Warm up the SQ1 at 37 C water bath 7 Under cool ambient conditions crystals may form in some of the buffers This is normal and the bottle should be warmed to re dissolve the salt Procedure 1 Add 15 mL of SQ 1 Buffer to a 50mL Centrifuge Tube Grind fresh plant tissue finely in liquid nitrogen with a porcelain mortar and pestle Weigh 500 mg frozen ground tissue and transfer to the 50 ml centrifuge tube containing the 15 mL of SQ1 Buffer 2 Homogenize the sample by vortexing or using 5 10 times strokes with a microfuge tube pestle 3 Add 5 mL SQ2 Buffer to the cell lysate 4 Invert the tube gently 10 15 times and incubate on ice for 5 minutes 5 Centrifuge at gt 4 000 x g at room temperature for 15 minutes 6 Carefully transfer the cleared supernatan
4. rifuge capable at least 4 000 x g 7 70 ethanol m 100 Isopropanol m 15 mL Centrifuge TubeS m waterbath or incubator Before starting 7 Preheat a water bath to 65 C Warm up the SQ1 at 37 C water bath 7 Under cool ambient conditions crystals may form in some of the buffers This is normal and the bottle should be warmed to re dissolve the salt Procedure 1 Add 6 mL of SQ 1 Buffer to a 15 mL Centrifuge Tube Grind fresh plant tissue finely in liquid nitrogen with a porcelain mortar and pestle Weigh 100 200 mg frozen ground tissue and transfer to the 15 ml microfuge tube containing the 6 mL of SQ1 Buffer 2 Homogenize the sample by vortexing or using 5 10 times strokes with a microfuge tube pestle 3 Add 2 mL SQ2 Buffer to the cell lysate 4 Invert the tube gently 10 15 times and incubate on ice for 5 minutes 5 Centrifuge at gt 4 000 x g at room temperature for 15 minutes 6 Carefully transfer the cleared supernatant into a new 15 ml microtube Note Do not disturb the pellet because it contains proteins 7 Add 20 uL of Rnase A to the sample Mix the sample by vortexing for 5 seconds 8 Add an equal volume of isopropanol to the sample Invert the tube 10 20 times 10 11 12 13 Centrifuge at 4 000 xg at room temperature for 15 minutes The DNA will form a small translucent pellet Carefully remove the supernatant Add 6 mL 70 ethanol to the tube Wash the DNA pellet by inverting the tube 5 10 t
5. t into a new 50 ml microtube Note Do not disturb the pellet because it contains proteins 7 Add 50 uL of Rnase A to the sample Mix the sample by vortexing for 5 seconds 8 Add an equal volume of isopropanol to the sample Invert the tube 10 20 times 10 11 12 13 Centrifuge at 4 000 xg at room temperature for 15 minutes The DNA will form a small translucent pellet Carefully remove the supernatant Add 15 mL 70 ethanol to the tube Wash the DNA pellet by inverting the tube 5 10 times Centrifuge at 4 000 x g at room temperature for 15 minutes Carefully remove the supernatant and drain tube on a clean absorbent paper Air dry the DNA pellet for 10 minutes Add 1 5 ml of Buffer EB to the tube Vortex the tube for 10 seconds Allow the DNA to re hydrate at 65 C for at least 30 minutes Store the sample at 80 C Trouble Shooting Problem Likely Cause Suggestions Low nucleic acid yield Incomplete disruption and homogenization of See cell lysis and homogenization instruction If the lysate is too viscous a mechanic homogenizer may be needed DNA degraded Make sure the sample is during sample properly stored and make sure storage the samples are processed immediately after collection or removal from storage Loss of DNA Be careful not to lose the DNA pellet during pellet during the operation operation Ethanol carryover Make sure the ethanol is completely remo
6. trogen with a porcelain mortar and pestle Weigh 10 25 mg frozen ground tissue and transfer to the 1 5 ml centrifuge tube containing the 750ul of SQ1 Buffer 2 Homogenize the sample by vortexing for 30 seconds 3 Add 250ul SQ2 Buffer to the cell lysate 4 invert the tube gently 10 15 times and incubate on ice for 5 minutes 5 Centrifuge at gt 13 000 xg at room temperature for 5 minutes 6 Carefully transfer the cleared supernatant into a new 1 5 ml microtube Note Do not disturb the pellet because it contains proteins 7 Add 2uL of Rnase A to the sample Mix the sample by vortexing for 5 seconds 8 Add an equal volume of isopropanol to the sample Invert the tube 10 20 times 10 11 12 13 Centrifuge at gt 13 000 x g at room temperature for 5 minutes The DNA will form a small translucent pellet Carefully remove the supernatant Add 750ul 70 ethanol to the tube Wash the DNA pellet by inverting the tube 5 10 times Centrifuge at gt 13 000 x g at room temperature for 5 minutes Carefully remove the supernatant and drain tube on a clean absorbent paper Air dry the DNA pellet for 10 minutes Add 100 ul of Buffer EB to the tube Vortex the tube for 10 seconds Allow the DNA to re hydrate at 65 C for at least 30 minutes Store the sample at 80 C B 100 200 mg SQ Plant DNA protocol Material and Equipments supplied by User Have the following reagents and supplies ready before starting procedure m Cent
7. ved before DNA rehydration 10
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