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Polysciences PEI溶液配制說明書
——線性聚乙烯亞胺, #23966,1mg/ml轉染試劑的配制
轉染試劑線性PEI溶液的配制
聚乙烯亞胺(Polyethylenimine,PEI)是聚乙烯亞胺是一種具有較高的陽離子電荷密度的有機大分子,每相隔二個碳個原子,即每“第三個原子都是質子化的氨基氮原子,使得聚合物網絡在任何pH下都能充當有效的“質子海綿”(proton sponge)體。PEI,可用作轉染試劑,它可以縮聚DNA分子形成顆粒并轉染真核細胞。有實驗證明,分子量為25000的線性PEI即Polysciences 23966轉染效率zui高。
下面將主要介紹polysciences 線性PEI(#23966)的產品特性及其溶液的配制過程。
1.產品特性:
產品名稱:線性聚乙烯亞胺(PEI)
CAS號 | 9002-98-6,26913-06-4 | 分子式 | (CH2CH2NH)n | 分子量 | 25000 |
熔點 | 73-75°C | 外觀 | 白色或黃色固體 | 結構式 | |
溶解性 | 溶于熱水,低pH值的冷水,甲醇和乙醇;不溶于:苯,Ethyl ether 和丙酮 | 儲存 | 室溫 |
2.溶液配制
此指導說明書是經polysciences公司研究,測試及其客戶反饋的后編寫的。下面推薦一種簡單適用而的方法,講述如何配制濃度為1mg/mL的轉染試劑的配制。
材料:
1g #23966、1L 純水(Milli-Q®純水,注射用水(WFI),或類似的生物級水)、12 M鹽酸、10 M氫氧化鈉、一次性0.1-0.2µm PES真空無菌過濾器、無菌高聚乙烯或聚丙烯試劑瓶。
器材:
1 L 玻璃燒杯、1 L玻璃量筒、核準pH計、2支1 mL塑料移液管、托盤、聚四氟乙烯(PTFE)攪拌棒、真空泵
配制過程:
1g #23966——900mL水溶解——攪拌、加熱(60-80°C)——加HCl調pH到2.0左右——蓋上燒杯,攪拌3h(直至粉末*溶解)——冷卻至室溫——加NaOH調pH到6.9-7.1——移液到量筒,加水補足至1L——真空過濾消毒——分裝,-20°C保存。
備注:加熱或加酸都有助于PEI溶解。當然酸可能不用加那么多,詳細用量可試加一點攪拌看看,足夠溶解就行,不一定需要加到pH那么低。
試劑儲存:
冷凍部分溶液可在-20°C保存長達一年;部分解凍的溶液可在4°C保存2周,不可反復凍融。
粉末儲存:
未開封的粉末有效期兩年??稍谑覝叵卤4嬷敝潦褂?。
參考文獻:
1.聚乙烯亞胺(PEI)、線性 (1 mg/mL). Cold Spring Harbor Protocols 2008, pdb.rec11323–pdb.rec11323 (2008). doi:10.1101/pdb.rec11323.
Catalog# 23966 –Polyethylenimine, Linear, MW 25000 (PEI 25K)
Transfection Reagent Preparation and Storage Recommendations
These guidelines are our recommendations based off research, testing, and customer feedback. There are alternative methods available, but we suggest following these recommendations for easy adoption and consistently high performance. These methods are adapted from Cold Spring Harbor Protocols [1], an excellent secondary resource for transient transfection literature.
Reagent Preparation (1 mg/mL)
Materials:
1. 1g PEI 25K (Polysciences Catalog# 23966).
2. 1L Milli-Q® water, water for injection (WFI), or comparable biological-grade water
3. 12 M Hydrochloric Acid (HCl)
4. 10 M Sodium Hydrochloride (NaOH)
5. Disposable 0.1-0.2µm PES vacuum sterile-filter.
6. Sterile HDPE or polypropylene storage vials for reagent aliquots.
Equipment
1. 1L glass beaker
2. 1L glass graduated cylinder
3. Calibrated pH meter
4. 2 x disposable 1mL plastic pipettes
5. Stir plate
6. PTFE coated stir bar
7. Vacuum pump
Method:
1. Dispense 1g of PEI 25K into beaker and suspend in 900 mL of water.
2. Add stir bar and set stirring to produce a small vortex.
3. Add hydrochloric acid dropwise until pH is < 2.0.
4. Cover top of beaker and stir for up to 3 hours until powder fully dissolves.
5. Add sodium hydroxide dropwise until pH is 6.9-7.1.
6. Transfer solution to graduated cylinder and add water until the total volume is 1L.
7. Sterile-filter through vacuum membrane.
8. Aliquot as desired and store at -20°C.
Reagent Storage
Frozen aliquots can be stored at -20°C for up to one year. Aliquots can be thawed and kept at 4°C for up
to two weeks, but should never be re-frozen.
Powder Storage
Unopened powder has a shelf life of two years from date of receipt. It can be kept at room temperature
until use.
References
1. Polyethylenimine (PEI), linear (1 mg/mL). Cold Spring Harbor Protocols 2008, pdb.rec11323-pdb.rec11323 (2008). doi:10.1101/pdb.rec11323
Support
We’re here to help! Our technical staff can troubleshoot most common and not-so-common issues you
may have. Send us an at info@polysciences.com and we will get back to you, typically within one
business day.
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