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名称:组织细胞DNA提取和扩增试剂盒
品牌:ACTGene 型号:组织细胞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:26418
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简介:Tissue 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. Tissue Cell DNA Extraction from Mouse Tails, Animal, Tissues, Hair, or Saliva
1. Pipette 100ul of Extraction Solution into a microcentrifuge tube or a multiwell plate well. Add 25ul 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 4:1 up to 2 hours before use.
2a. For Fresh or Frozen Mouse Tails: Rinse the scissors and forceps in 70% ethanol prior to use and between different samples. Place a 0.5 - 1 cm piece of mouse tail tip (cut end down) into the solution. Mix thoroughly by vortexing or pipetting. Ensure that the mouse tail is in solution.
Note: For fresh mouse tails, perform extractions within 30 minutes of snipping the tail.
2b. For Animal tissues: Rinse the scissors or scalpel and forceps in 70% ethanol prior to use and between different samples. Place a 2-10 mg piece of tissue into the solution. Mix thoroughly by vortexing or pipetting. Ensure that the tissue is in the solution.
2c. For Hair Shafts: Rinse the scissors and forceps in 70% ethanol prior to use and between different samples. Trim excess off of the hair shaft leaving the root and place sample (root end down) into solution. Only one hair shaft, with root, is required per extraction.
2d. For Saliva: Pipette 10ul of saliva into the solution. Mix thoroughly by vortexing or pipetting.
2e. For Saliva Dried on Card:Pipette 50ul of saliva onto collection card and allow the card to dry. Rinse the punch in 70% ethanol prior to use and between different samples. Punch a disk (preferably 1/8 inch or 3 mm) out of the card from the area with the dried saliva sample. Place disk into the solution. Tap tube or plate on hard surface to ensure disk is in solution for incubation period.
3. Incubate samples at room temperature for 10 minutes.
4. Incubate samples at 95℃ for 3 minutes.
Note: Tissues will not be completely digested at the end of the incubations. This is normal and will not affect performance.
5. Add 100ul of Neutralization Solution to sample and mix by vortexing.
6. Use immediately in PCR or store the neutralized tissue extract at 4℃. Continue with Section C, Step 1.
Note: Prior to storage, remove the undigested tissue by centrifugation and transfer the extracts to new tubes or wells.
B. Cell DNA Extraction for Buccal Swabs
1. Collect buccal cells on swab and allow the swab to dry for 10 to 15 minutes.
2. Pipette 200ul of Extraction Solution into a 1.5 ml microcentrifuge tube. Add 50ul of Preparation Solution to the tube 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 4:1 up to 2 hours before use.
3. Place dried buccal swab into the solution and incubate at room temperature for 1 minute.
4. Twirl swab in solution 10 times and then twirl swab firmly against the side of the tube to remove excess solution into the tube. Discard the swab. Close the tube and vortex briefly.
5. Incubate samples at room temperature for 10 minutes.
6. Incubate samples at 95℃ for 3 minutes.
7. Add 200ul of Neutralization Solution to sample and mix by vortexing.
8. Use immediately in PCR or store the neutralized extract at 4℃ . Continue with Section C, Step 1.
Note: Prior to storage, remove the undigested cells 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 leaf disk 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 tissue extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction Solution : Neutralization Solution to compensate the volume of tissue 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 3ml in 5ml bottle 24ml in 30ml bottle 250ml in 250ml or
2x125ml bottles
Preparation Solution 0.35ml in 1.5 ml vial 3ml in 5ml bottle 30ml in 30ml bottle
Neutralization Solution 3ml in 5ml bottle 24ml in 30ml bottle 250ml in 250ml or
2x125ml bottles
Hot-Start Taq Blue Mastermix 0.15ml in 1.5ml vial 1.2ml in 1.5ml vial 12ml in 15ml conical tube