کیت سنتز سی دی اِن آ | cDNA synthesis Kit

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عملکرد عالی
قابلیت سنتز cDNA از RNA هایی با غلظت های پایین (1ng)
قابلیت استفاده از پرایمرهای Oligo dt و Random hexamer به صورت همزمان
سنتز به واسطه ترانس کریپتاز معکوس بسیار کارآمد MMLV
cDNA حاصل قابل استفاده در کلیه واکنش های پایین دستی
امکان سنتز cDNA از میکروRNA در صورت طراحی پرایمر اختصاصی توسط کاربر

برند	یکتا تجهیز آزما YTA

شناسه محصول: YT4500 دسته: RT-PCR, آنزیم ها و مستر میکس, محصولات

توضیحات

کیت سنتز cDNA synthesis Kit | cDNA

کیت سنتز cDNA شرکت یکتا تجهیز آزما یک کیت کامل است که شامل تمام اجزای مورد نیاز برای انجام یک سنتز cDNA کارآمد از الگوهای RNA می باشد.

این اجزا شامل یک آنزیم رونویسی معکوس (RT) ، dNTPs، بافر واکنش ، پرایمر و مهارکننده‌های RNase (RNase inhibitor) می باشد که RNase Inhibitor موجود در این کیت یک مهارکننده قوی RNaseها است.

علاوه بر پرایمر رندوم هگزامر موجود (Random hexamer) که به همه RNA های موجود در واکنش اجازه می دهد به عنوان الگو استفاده شوند ، یک پرایمر الیگو (Oligo dt) نیز برای سنتزcDNA از دم پلی A (Poly A tail) و به منظور افزایش انعطاف پذیری در کاربرد درکیت قرار داده شده است.

این کیت حاوی آنزیم رونویسی معکوس M-MLV Reverse Transcriptase (RT) با ویژگی عدم وجود RNaseH (RNase H- ) و نیز پایداری در برابر حرارت است.

وجود مواد مورد نیاز با کیفیت به همراه دستورالعمل بهینه ارائه شده ، منجر به افزایش بازده سنتز cDNA شده است.

این کیت به دلیل عملکرد قوی ، سهولت استفاده و سازگاری با واکنش های مختلف پایین دست وابسته به cDNA ، یک گزینه مناسب جهت cDNA synthesis می باشد.

کاربرد :

سنتزcDNA از mRNA
سنتزcDNA از میکروRNA در صورت طراحی پرایمر اختصاصی توسط کاربر

شرایط نگهداری:

در دمای 20- نگهداری شود

دستورالعمل ها

دانلود دستورالعمل کیت سنتز سی دی اِن آ | cDNA synthesis Kit

سوالات متداول

در صورت استفاده از هر دو پرایمر الیگو و رندوم هگزامر دمای واکنش را چه دمایی بگذاریم؟

با توجه به اینکه دمای مورد نیاز برای الیگو dt دمای 42 درجه سانتی گراد و دمای مورد نیاز برای رندوم هگزامر دمای 37درجه سانتی گراد می باشد بهتر است هنگام استفاده از هر دو پرایمر در یک واکنش، دمای 40 درجه سانتی گراد جهت انجام واکنش انتخاب شود.

مراحل سنتز cDNA شامل دو مرحله است که در هر مرحله یک تعدادی از مواد کیت با هم مخلوط می شوند، سوال اینجاست آیا این میکس های دو مرحله با هم مخلوط می شوند؟

پاسخ بله است، بر اساس پروتوکل کیت سنتز cDNA شرکت یکتا تجهیزآزما ابتدا در مرحله اول پرایمر، آب و نمونه را در یک ویال ریخته و به مدت 5دقیقه در دمای 70 درجه قرار می دهیم دراین فاصله زمانی یک میکروتیوب دیگر بردارید و بافر،dNTP ،RNasin و MMLV را بر اساس مقادیر گفته شده به هم اضافه میکنیم تا مخلوط دوم حاصل شود.پس از اتمام زمان 5دقیقه مخلوط اول را به مخلوط دوم اضافه کنید و در در انتها واکنش نهایی را در ترموسایکلر به مدت یک ساعت قرار دهید.

در کیت سنتز cDNA آیا می توان از دو پرایمر Oligodt و Random hexamer همزمان در واکنش سنتز استفاده کرد؟

پرایمر Random hexamer و پرایمر اولیگو dT دو نوع پرایمر هستند که در سنتز cDNA استفاده می شوند. Random hexamer از مخلوطی از اولیگونوکلئوتیدها تشکیل شده است که در طول تولید cDNA بع طور تصادفی به تمام مناطق RNA متصل می شود و یک مخزن cDNA حاوی طول های مختلف cDNA حاصل می شود. ، در حالی که پرایمر الیگو dT یک توالی تک رشته ای از 12 تا 18 نوکلئوتیدی است که dT به دنباله های polyA پیام رسان (mRNA) متصل و سنتز DNA مکمل (cDNA) را هدایت می کند. بنابراین، این تفاوت اصلی بین پرایمرهای تصادفی و الیگو dT است. علاوه بر این، پرایمرهای الیگو dT برای رونویسی معکوس تمام طول RNA با دم پلی (A) به کار بسته می شوند، در حالی که Random hexamer برای رونویسی معکوس اکثر گونه‌های RNA، از جمله RNA تخریب‌شده، RNA فاقد دم پلی (A) و RNA حاوی ساختارهای ثانویه کارایی دارد. بنابراین می توان از هر دو پرایمر به صورت همزمان در دمای 40 درجه سانتی گراد استفاده کرد.

آیا می توان برای سنتز میکروRNA از این کیت های سنتز cDNA استفاده کرد؟

بله، در صورتی که کاربر یک پرایمر اختصاصی برای سنتز cDNA ازمیکروRNA اختصاصی خود طراحی کند می تواند از سایر اجزای موجود در کیت جهت سنتز استفاده کند.

در صورت استفاده همزمان از دو پرایمر اولیگو و رندوم هگزامر در سنتز cDNA، دمای واکنش را روی چه درجه ای تنظیم کنیم؟

هنگام انجام سنتز cDNA با استفاده از کیت سنتز cDNA ؛ در صورت استفاده همزمان از دو پرایمر الیگو و رندام هگزامر دمای واکنش را روی درجه تنظیم کنید.

در صورت استفاده ازپرایمر لوپ ( (stem loop جهت micro RNA ، از چه دمایی برای واکنش استفاده کنیم ؟

تنطیم دمای پرایمر لوپ بستگی به خود پرایمر مورد استفاده دارد.

حداقل میزان Total RNA برای سنتز cDNA چقدر باید باشد؟

چنانچه کیفیت RNA خوب باشد از مقادیر پایین هم cDNA قابل سنتز هست،براساس پروتکل از 1 نانوگرم میشود سنتز انجام داد،اما به طور کلی اگر مثلا 50 نانوگرم از RNA داشته باشید میتوانید بطور مثال از 5 لاندا نمونه استفاده کنید.

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