Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids

Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.

Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5' ends and insert sequence at the 3' ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), beta-d-glucuronidase (gusA), and beta...

Repeatable Construction Method for Engineered Zinc Finger Nuclease Based on Overlap Extension PCR and TA-Cloning

Zinc finger nuclease (ZFN) is a useful tool for endogenous site-directed genome modification. The development of an easier, less expensive and repeatedly usable construction method for various sequences of ZFNs should contribute to the further widespread use of this technology. Here, we establish a novel construction method for ZFNs. Zinc finger (ZF) fragments were synthesized by PCR using shor...

Rapid and reliable cloning of PCR products.

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method

Background: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. Objectives: We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR.   Materials and Methods: Genomic DNA from Bacillus thermo...

طراحی و ایجاد موتاسیون در ژن wbk A بروسلا آبورتوس S19 به روش Overlap Extension PCR

زمینه مطالعه: ایجاد موتاسیون در جایگاه اختصاصی می‌تواند یکی از روش‌های کارآمد جهت بررسی ویژگی و خواص تنظیمی ژن‌های گوناگون باشد. ﺑﺮوﺳﻠﻮز از ﻣﻬﻢ ﺗﺮﯾﻦ ﺑﯿﻤﺎریهای ﻋﻔﻮﻧﯽ ﻣﺸﺘﺮک ﺑﯿﻦ اﻧﺴﺎن و دام اﺳﺖ ﻛﻪ ﻣﻨﺠﺮ ﺑﻪ بروز ﺿﺮرهاى اﻗﺘﺼﺎدى ﻓﺮاواﻧﻰ ﻣﻰ ﺷﻮد. ﺑﻨﺎﺑﺮاﯾﻦ ﺷﻨﺎﺳﺎﯾﯽ ﻋﻮاﻣﻞ پاتوژن و اﯾﻤﻨﯽ زا در جنس ﺑﺮوﺳﻼ ﺑﻪ ﻋﻨﻮان راهگشای ﺟﻬﺖ ﮐﻨﺘﺮل اﯾﻦ ﻣﻌﻀﻞ ﺑﻬﺪاﺷﺘﯽ ﻣﻄﺮح ﻣﯽ‌ﺑﺎﺷﺪ. هدف: با توجه به اهمیت جهش هدفدار در شناسایی ساخ...