暂无图片
名称:种子细胞DNA提取和扩增试剂盒
品牌:ACTGene 型号:种子细胞DNA提取和扩增试剂盒
产地:TW 新旧:0
价格:0 数量:2
是否促销:否
浏览次数:6009
加入收藏 |
简介:Seed Cell DNA Extraction & Amplification Kit
Storage : The kit should be stored at -20℃.
Procedure :
All steps are performed at room temperature unless otherwise noted.
A. Seeds Grinding
1. Grind using a bead mill.
a. Place 1 seed into each well of a 2 ml square well block.
b. Pipette the following volumes of water into the well:
800ul for soybean or similar sized seeds.
600ul for cotton or similar sized seeds.
200ul for canola, sorghum, wheat, or similar sized seeds.
100ul for Arabidopsis or similar sized seeds.
c. Place a 4 mm stainless steel grinding ball in each well of the 2 ml 96 square well block and cover with sealing mat. Place block in the bead mill and shake at 1,500 rpm for 10 minutes. Continue with Section B, Step 1.
2. Grind using a plastic pestle
a. Place 1 seed into a 1.5 ml microcentrifuge tube.
b. Pipette the following volumes of water into the tube:
800ul for soybean or similar sized seeds.
600ul for cotton or similar sized seeds.
200ul for canola, sorghum, wheat, or similar sized seeds.
100ul for Arabidopsis or similar sized seeds.
c. Incubate the seed for 1 hour at 55℃.
d. Grind hydrated seeds in tube using a plastic pestle. Continue with Section B, Step 1.
3. Grind using liquid nitrogen.
a. Use a mortar and pestle to grind seed into a fine powder in liquid nitrogen.
b. Transfer between 5-100 mg of ground seed material into a pre-weighed 1.5 ml microcentrifuge tube. Record the mass of the transferred seed material.
c. Pipette 4uL of water for every mg of transferred ground seed material into the tube and vortex to mix well. Continue with Section B, Step 1.
B. Seeds DNA Extraction
1. Pipette 45ul of Extraction Solution into a microcentrifuge tube or a multiwell plate well. Add 5ul of Preparation Solution to the tube or well and pipette up and down to mix.
Note: If several extractions will be performed, sufficient volumes of Extraction Solution and Preparation Solution may be mixed in a ratio of 9:1 up to 2 hours before use.
2. Pipette 5ul of the ground seed suspension from Section A into the tube or well and vortex to mix.
3. Incubate samples at 55℃ for 10 minutes.
4. Incubate samples at 95℃ for 3 minutes.
5. Add 50ul of Neutralization Solution to each sample and mix by vortexing.
6. Use immediately in PCR or store the neutralized seed extract at 4℃ or. Continue with Section C, Step 1.
Note: Prior to storage, remove the undigested seed by centrifugation and transfer the extracts to new tubes or wells.
C. PCR Amplification
Taq mastermix, Hot-Start Taq mastermix, Taq Blue mastermix, or Hot-Start Taq Blue mastermix can be used for the following PCR amplification steps. Typical final primer concentrations are approximately 0.4uM each. Primer concentrations and thermal cycling parameters should be optimized depending on the system.
1. Add 10ul of PCR mastermix (2x), 4ul of seed extract, and proper amount of primers to a thin-walled PCR microcentrifuge tube or plate. Bring the total volume to 20ul with PCR grade water. Gently mix well.
Note: If less than 4ul of seed extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction Solution : Neutralization Solution to compensate the volume of seed extract to 4ul.
2. Perform thermal cycling using optimized amplification parameters.
3. Load the amplified DNA onto an agarose gel after the PCR is completed.
Kit Specification:
Reagents 10-rxn 100-rxn 1000-rxn
Extraction Solution 1ml in 1.5ml vial 6ml in 10ml bottle 60ml in 65ml bottle
Preparation Solution 0.1ml in 1.5ml vial 0.9ml in 1.5ml vial 9ml in 10ml bottle
Neutralization Solution 1ml in 1.5ml vial 6ml in 10ml bottle 60ml in 65ml bottle
Hot-Start Taq Blue Mastermix 0.15ml in 1.5ml vial 1.2ml in 1.5ml vial 12ml in 15ml conical tube