Online English Summarizer tool, free and accurate!
How to Design Primers for PCR and quantitative real time PCR (qPCR) Tips for primer design:
Keep the melting temperatures (Tm) of each primer pair within 2?C of one another.Cytosine and guanine have stronger binding affinity than adenine and thymine and repeats of more than 4 G or C can bind to many places in the genome with high affinity.A GC content between 35% and 65% without long stretches (> 4 bases) of the same nucleotide will ensure enough sequence complexity for optimal primer specificity.Amplicons between 70-140 base pairs are generally long enough to allow the design of two efficient primers and a probe (if a TaqMan-based assay is desired) for qPCR assays.Having a similar Tm between primers ensures that the forward and reverse primers will be bound to their complementary DNA strands at the same time, reducing the chance that the primer with the highest Tm will bind to nonspecific DNA sequences.If these repeats are at the 3' end of the primer, DNA polymerase can extend amplicons in off-target locations, which can ultimately decrease the PCR efficiency.
How to Design Primers for PCR and quantitative real time PCR (qPCR)
Tips for primer design:
Keep the melting temperatures (Tm) of each primer pair within 2°C of one another. The Tm can be approximately calculated by the formula Tm = (A+T) x 2 + (G+C) x 4, however, more precise, and elaborate Tm calculation tools are available online. Having a similar Tm between primers ensures that the forward and reverse primers will be bound to their complementary DNA strands at the same time, reducing the chance that the primer with the highest Tm will bind to nonspecific DNA sequences.
Use an annealing temperature (Ta) 3-5°C below the melting temperature. The annealing temperature is the temperature at which the primers will bind to a new template strand. This needs to be lower than the Tm so that the primers can efficiently bind to the target, but not too low that the primers bind to nonspecific targets.
Keep primers between 18-22 base pairs long. This primer length is long enough to ensure binding specificity while also short enough to keep the Tm within an appropriate range.
Design primers with a GC content of 35-65%. A GC content between 35% and 65% without long stretches (> 4 bases) of the same nucleotide will ensure enough sequence complexity for optimal primer specificity.
Minimize G/C repeats, especially at the 3’ end of the primer. Cytosine and guanine have stronger binding affinity than adenine and thymine and repeats of more than 4 G or C can bind to many places in the genome with high affinity. If these repeats are at the 3’ end of the primer, DNA polymerase can extend amplicons in off-target locations, which can ultimately decrease the PCR efficiency.
Limit amplicon length to 140 base pairs. Amplicons between 70-140 base pairs are generally long enough to allow the design of two efficient primers and a probe (if a TaqMan-based assay is desired) for qPCR assays. Longer amplicons are possible, although it may be necessary to adjust the thermal cycler protocol to allow for complete elongation of the new strand. Additionally, if the sample contains heavily fragmented DNA, it is best to keep amplicons smaller to increase the likelihood of having intact target sequences for primer binding.
Summarize English and Arabic text using the statistical algorithm and sorting sentences based on its importance
You can download the summary result with one of any available formats such as PDF,DOCX and TXT
ٌYou can share the summary link easily, we keep the summary on the website for future reference,except for private summaries.
We are working on adding new features to make summarization more easy and accurate
لقد حقق القسم إنجازات متعددة تعكس دوره المحوري في مواجهة تحديات التغيرات المناخية في القطاع الزراعي....
1. قوة عمليات الاندماج والاستحواذ المالية في المشهد الديناميكي للأعمال الحديثة، ظهرت عمليات الاندماج...
اﻷول: اﻟﺒﺤﺚ ﻋﻠﻰ ﺗﺸﺘﻤﻞ ﺗﻤﮭﯿﺪﯾﺔ ﻣﻘﺪﻣﮫ ﺳﻨﻀﻊ اﻟﻤﺒﺤﺚ ھﺬا ﻓﻲ ﺳﺘﻜﻮن ﺧﻼﻟﮭﺎ ﻣﻦ واﻟﺘﻲ اﻟﻌﻼﻗﺔ ذﻟﺒﻌﺾ ھﺎﻌﻠﻮم ﻔﺎت ...
الوصول إلى المحتوى والموارد التعليمية: تشكل منصات وسائل التواصل الاجتماعي بوابة للدخول إلى المحتوى ...
ـ أعداد التقارير الخاصه بالمبيعات و المصاريف والتخفيضات و تسجيل الايرادات و المشتريات لنقاط البيع...
وهي من أهم مستحدثات تقنيات التعليم التي واكبت التعليم الإلكتروني ، والتعليم عن والوسائط المتعدد Mult...
كشفت مصادر أمنية مطلعة، اليوم الخميس، عن قيام ميليشيا الحوثي الإرهابية بتشديد الإجراءات الأمنية والر...
أولاً، حول إشعياء ٧:١٤: تقول الآية: > "ها إن العذراء تحبل وتلد ابنًا، وتدعو اسمه عمانوئيل" (إشعياء...
يفهم الجبائي النظم بأنّه: الطريقة العامة للكتابة في جنس من الأجناس الأدبية كالشعر والخطابة مثلاً، فط...
أعلن جماعة الحوثي في اليمن، اليوم الخميس، عن استهداف مطار بن غوريون في تل أبيب بصاروخ باليستي من نوع...
اهتم عدد كبير من المفكرين والباحثين في الشرق والغرب بالدعوة إلى إثراء علم الاجتماع وميادينه، واستخدا...
وبهذا يمكن القول في هذه المقدمة إن مصطلح "الخطاب" يعدُّ مصطلحًا ذا جذور عميقة في الدراسات الأدبية، ح...