Primer sets, reagents, kits, applications and identification methods for identifying apical papilla stem cells
A technology of papillae stem cells and primer sets, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of high instrument dependence and high cost, and achieve high sensitivity, short detection cycle and repeatability Good results
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Embodiment 1
[0095] 1. Total RNA extraction
[0096] Before the experiment, the operating environment was treated with an RNA inhibitor. In the following operations, wear a mask, take the conical flask containing the pipette tip, centrifuge tube, etc. horizontally, and then cover the bottle immediately to avoid environmental pollution.
[0097] 1) Pour off the medium in the petri dish / flask, wash 2-3 times with PBS, add 0.7mL Trizol solution (red), blow off the cells attached to the petri dish / flask with a gun, and transfer directly to a 1.5mL centrifuge In the tube, add 0.5 mL of Trizol to the culture dish to rinse the culture dish to harvest the remaining incompletely blown cells, transfer to the same centrifuge tube, and after shaking, let stand at room temperature for 5 min.
[0098] 2) Add 200 μL of chloroform directly, mix by vortexing for 15 s, and place at room temperature for 2-3 min (adding chloroform can fully separate proteins, DNA and lipids).
[0099] 3) Centrifuge at 12000...
Embodiment 2
[0118] Add 1 mL of lysis buffer to lyse cells in a petri dish, resuspend the cell pellet, stand on ice for 30 min and centrifuge at 10,000 × g for 10 min at 4°C. Carefully separate the supernatant and transfer to a new tube. Be careful not to pipette into the upper fat layer and the lower sediment layer. The protein concentration of the supernatant was determined by the BCA protein assay kit.
[0119] Transfer 300 μg of protein to a new microcentrifuge tube and adjust to a final volume of 100 μL with 100 mM TEAB. Add 1.5 [mu]L of 1M DTT, 56[deg.]C for 1 hour. Add 8 μL of 1M IAA and protect from light at room temperature for 45 min. Add 50mM NH to the reaction solution 4 HCO 3 2.5 μg trypsin was added. Samples were digested overnight at 37°C.
[0120] To the digested samples, FA was added to a final concentration of 0.1% to acidify the samples. Desalting was performed in C18 waters. Proceed as follows:
[0121] Activation: wash twice with acetonitrile, 1 mL each time;...
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