scholarly journals Straightforward Procedure for Laboratory Production of DNA Ladder

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Vo Thi Thuong Lan ◽  
Pham Thi Thanh Loan ◽  
Pham Anh Thuy Duong ◽  
Le Thi Thanh ◽  
Ngo Thi Ha ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Tandem Repeat ◽  
Restriction Site ◽  
Restriction Enzymes ◽  
Dna Fragments ◽  
Repeated Dna ◽  
Dna Ladder ◽  
Time Saving ◽  
Partial Digestion ◽  
Unique Restriction

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

scholarly journals Construction of DNA ladder for determination of small size DNA fragments

2021 ◽  
Vol 19 (3) ◽  
pp. 539-545
Author(s):  
Vo Thi Thuong Lan ◽  
Le Thi Thanh
Keyword(s):  
Molecular Biology ◽  
Restriction Enzyme ◽  
Dna Markers ◽  
Dna Marker ◽  
Dna Fragments ◽  
Dna Ladder ◽  
Time Saving ◽  
Pcr Product ◽  
Dna Fragment

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.

scholarly journals Preparation of DNA Ladder Based on Multiplex PCR Technique

2010 ◽  
Vol 2010 ◽  
pp. 1-3 ◽  
Author(s):  
Tian-Yun Wang ◽  
Li Guo ◽  
Jun-he Zhang
Keyword(s):  
Molecular Biology ◽  
Dna Marker ◽  
Dna Fragments ◽  
Dna Ladder ◽  
Hot Start ◽  
Target Dna ◽  
Touchdown Pcr ◽  
Lambda Dna ◽  
Molecular Weight Standard ◽  
Multiple Pcr

DNA molecular weight standard control, also called DNA marker (ladder), has been widely used in the experiments of molecular biology. In the paper, we report a method by which DNA marker was prepared based on multiple PCR technique. 100–1000 bp DNA fragments were amplified using the primers designed according to the 6631 ~ 7630 position of lambda DNA. Target DNA fragments were amplified using Touchdown PCR combined with hot start PCR, respectively, followed extracted by phenol/chloroform, precipitated with ethanol and mixed thoroughly. The results showed that the 100–1000 bp DNA fragments were successfully obtained in one PCR reaction, the bands of prepared DNA marker were clear, the size was right and could be used as control in the molecular biology experiment. This method could save time and be more inexpensive, rapid, simple when compared with the current DNA Ladder prepared means.

scholarly journals Engineering a Minimal Cloning Vector from a pUC18 Plasmid Backbone with an Extended Multiple Cloning Site

2018 ◽  
Author(s):  
Jens Staal ◽  
Wouter De Schamphelaire ◽  
Rudi Beyaert
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Essential Role ◽  
Building Blocks ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Unique Restriction ◽  
Plasmid Backbone

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

scholarly journals Identification of restriction enzyme in the FSHR gene of indonesian local cattle

2021 ◽  
Vol 888 (1) ◽  
pp. 012024
Author(s):  
P W Prihandini ◽  
A Primasari ◽  
M Luthfi ◽  
D Pamungkas ◽  
A P Z N L Sari ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Restriction Site ◽  
Follicle Stimulating Hormone ◽  
Restriction Enzymes ◽  
Future Studies ◽  
Fshr Gene ◽  
Pcr Rflp ◽  
Mapping Analysis ◽  
Pcr Products ◽  
Enzyme Mapping

Abstract The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.

scholarly journals Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination

BioTechniques ◽  
2009 ◽  
Vol 46 (7) ◽  
pp. 527-533 ◽  
Author(s):  
Stephan Noll ◽  
Gabriele Hampp ◽  
Hanna Bausbacher ◽  
Natalia S. Pellegata ◽  
Harald Kranz
Keyword(s):  
Copy Number ◽  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Unique Restriction Site ◽  
Unique Restriction

scholarly journals A Simple and Efficient Method to Isolate LTR Sequences of Plant Retrotransposon

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Da-Long Guo ◽  
Xiao-Gai Hou ◽  
Xi Zhang
Keyword(s):  
Molecular Markers ◽  
Efficient Method ◽  
High Efficiency ◽  
Size Structure ◽  
Restriction Enzymes ◽  
Rnase H ◽  
Plant Genome ◽  
New Method ◽  
Tree Peony ◽  
Time Saving

Retrotransposons (RTNs) have important roles in the formation of plant genome size, structure, and evolution. Ubiquitous distributions, abundant copy numbers, high heterogeneities, and insertional polymorphisms of RTNs have made them as excellent sources for molecular markers development. However, the wide application of RTNs-based molecular markers is restricted by the scarcity of the LTR (long terminal repeat) sequences information. A new, simple, and efficient method to isolate LTR sequences of RTNs was presented based on the degenerate RNase H nested primers and PPT (polypurine tract) primer of RTNs in tree peony. This method combined the characteristics and advantages of high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), annealing control primer (ACP) system, and suppression PCR method. Nineteen LTR sequences were isolated using this new method in tree peony and the applicability of the LTR sequences based markers was validated by further SSAP analysis. The results showed that the new method is simple, of low-cost, and highly efficient, which is just conducted by three rounds of PCR and does not need any restriction enzymes and adapters, much less the hybridizations. This new method is rapid, economical, and cost- and time-saving, which could be easily used to isolate LTR sequences of RTNs.

scholarly journals Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site

Microbiology ◽  
1995 ◽  
Vol 141 (10) ◽  
pp. 2425-2432 ◽  
Author(s):  
E. Jumas-Bilak ◽  
C. Maugard ◽  
S. Michaux-Charachon ◽  
A. Allardet-Servent ◽  
A. Perrin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Restriction Site ◽  
Brucella Melitensis ◽  
Unique Restriction Site ◽  
Unique Restriction
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