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 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 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 Asosiasi Marka Genetik dengan Pertambahan Bobot Badan Sapi Madura di Pamekasan

2017 ◽  
Vol 6 (1) ◽  
pp. 42
Author(s):  
Suyadi Suyadi ◽  
Isnaini Isnaini ◽  
Rahayu Rahayu ◽  
Y. Nurpah
Keyword(s):  
Growth Rate ◽  
Blood Sample ◽  
Dna Markers ◽  
Dna Polymorphism ◽  
Dna Marker ◽  
Efficient Tool ◽  
Salting Out ◽  
Key Words Dna ◽  
Pcr Product ◽  
Gh Gene

<p>Characteristic of DNA markers may be able to be used as useful and efficient tool to select merit animal from a population. In order to Madura cattle, growth rate is one of important traits should be considered. The aim of this study was to evaluate the association between the characteristic of DNA marker of candidate gene for growth hormone (GH) and growth rate of Madura cattle. A number of 10 female Madura cattle selected from 40 animals in average of about 18 months old were used as sample. The animal was weighed twice using electronic balance at the interval time of two months. At the simultaneous time to the animal weighing the blood sample was collected via vena jugularis 6 ml of each for DNA source. The blood sample was dropped ito polypropylene tubes containing EDTA for anti coagulant agent. DNA was isolated from leucocyte cells in the blood using salting out as standard method. PCR technique was used for amplifying the DNA using GH primer (forward: 5’-TAGGGAGGTGGAAAATGGA-3’ and reverse: 5’-GACACCTAGACAATGCG-3’). DNA polymorphism of GH gene was detected using RFLP technique by digesting the PCR-product DNA with HaeIII enzyme at position of GG*CC. The results showed that amplified DNA with this primer showed a single band of 450bp. Restriction of DNA with HaeIII enzyme resulted 4 haplotypes of uncut fragment , 2 fragments (2a and 2b), 3 fragments (3a and 3b) and 5 fragments at the position of 125bp, 200bp, 275bp and 450 bp. According to the data analysis, the non significant association was shown between specific genetic polymorphism and growth rate in this cattle.</p><p><em> </em></p><p>Key words: DNA marker, GH gene, growth rate, Madura cattle</p>

scholarly journals VALIDATION OF PCR-RFLP TESTING METHOD TO DETECT PORCINE CONTAMINATION IN CHICKEN NUGGET

2012 ◽  
Vol 12 (3) ◽  
pp. 302-307 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Winda Cahyaningtyas ◽  
Surajiman Surajiman ◽  
Istini Istini ◽  
Deni Pranowo
Keyword(s):  
Mitochondrial Dna ◽  
Rflp Analysis ◽  
Cytb Gene ◽  
Dna Fragments ◽  
Gene Fragment ◽  
Chicken Nugget ◽  
Testing Method ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Dna Fragment

PCR-RFLP technique to detect porcine contamination in chicken nugget has been developed and validated in this research. Various concentrations of pork were fortified during preparation of the nugget. DNA was then isolated from the nugget followed by PCR employed primers which targeted a 359 bp cytB gene fragment of mitochondrial DNA. For RFLP, the PCR product was digested by means of BamHI and BseDI enzymes. Cutting DNA fragments from nugget containing pork using BseDI enzyme produced DNA fragment with size 228 and 131 bp, while cutting with BamHI enzyme produce DNA fragments with sizes 244 and 115 bp. All of these fragments were not present in RFLP analysis of pork-free nugget. The method shows good specificity and precision and could detect porcine contamination in the nugget up to 5%. The method has been applied to test commercial nugget. Four brand of Halal-labeled commercial nugget as well as four brand of non labeled one gave negative porcine contamination.

scholarly journals Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis

2011 ◽  
Vol 78 (5) ◽  
pp. 1593-1595 ◽  
Author(s):  
Chun You ◽  
Xiao-Zhou Zhang ◽  
Y.-H. Percival Zhang
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Restriction Enzyme ◽  
Dna Fragments ◽  
Direct Transformation ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Pcr Product ◽  
Overlap Extension ◽  
General Restriction

ABSTRACTWe developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed inEscherichia coli[e.g., TOP10, DH5α, JM109, and BL21(DE3)] andBacillus subtilisfor obtaining chimeric plasmids.

scholarly journals Asosiasi Marka Genetik dengan Pertambahan Bobot Badan Sapi Madura di Pamekasan

2017 ◽  
Vol 6 (1) ◽  
pp. 42
Author(s):  
Suyadi Suyadi ◽  
Isnaini Isnaini ◽  
Rahayu Rahayu ◽  
Y. Nurpah
Keyword(s):  
Growth Rate ◽  
Blood Sample ◽  
Dna Markers ◽  
Dna Polymorphism ◽  
Dna Marker ◽  
Efficient Tool ◽  
Salting Out ◽  
Key Words Dna ◽  
Pcr Product ◽  
Gh Gene

<p>Characteristic of DNA markers may be able to be used as useful and efficient tool to select merit animal from a population. In order to Madura cattle, growth rate is one of important traits should be considered. The aim of this study was to evaluate the association between the characteristic of DNA marker of candidate gene for growth hormone (GH) and growth rate of Madura cattle. A number of 10 female Madura cattle selected from 40 animals in average of about 18 months old were used as sample. The animal was weighed twice using electronic balance at the interval time of two months. At the simultaneous time to the animal weighing the blood sample was collected via vena jugularis 6 ml of each for DNA source. The blood sample was dropped ito polypropylene tubes containing EDTA for anti coagulant agent. DNA was isolated from leucocyte cells in the blood using salting out as standard method. PCR technique was used for amplifying the DNA using GH primer (forward: 5’-TAGGGAGGTGGAAAATGGA-3’ and reverse: 5’-GACACCTAGACAATGCG-3’). DNA polymorphism of GH gene was detected using RFLP technique by digesting the PCR-product DNA with HaeIII enzyme at position of GG*CC. The results showed that amplified DNA with this primer showed a single band of 450bp. Restriction of DNA with HaeIII enzyme resulted 4 haplotypes of uncut fragment , 2 fragments (2a and 2b), 3 fragments (3a and 3b) and 5 fragments at the position of 125bp, 200bp, 275bp and 450 bp. According to the data analysis, the non significant association was shown between specific genetic polymorphism and growth rate in this cattle.</p><p><em> </em></p><p>Key words: DNA marker, GH gene, growth rate, Madura cattle</p>

scholarly journals Identifying and Mapping Two DNA Markers Linked to the Gene Conferring Resistance to Pea Enation Mosaic Virus

1995 ◽  
Vol 120 (5) ◽  
pp. 730-733 ◽  
Author(s):  
J. Yu ◽  
W.K. Gu ◽  
R. Provvidenti ◽  
N.F. Weeden
Keyword(s):  
Mosaic Virus ◽  
Dna Markers ◽  
Rapd Markers ◽  
Random Amplified Polymorphic Dna ◽  
The Other ◽  
Dna Fragments ◽  
Allele Specific ◽  
Dna Fragment ◽  
Pea Enation Mosaic Virus

Two random amplified polymorphic DNA (RAPD) markers linked to En, the gene conferring resistance to pea enation mosaic virus in pea, were identified and the DNA fragments were cloned and partially sequenced. Allele-specific associated primers for each cloned DNA fragment were developed and used in screening F2 populations. One marker, P256900, mapped very near Adh-1, about 6 cM from En. The other marker, B500400, was located about 8 cM from En on the same side as P256900.

Eco-friendly spectrophotometric methods for assessment of alfuzosin and solifenacin in their new pharmaceutical formulation; Green profile evaluation via eco-scale and GAPI tools

2020 ◽  
Vol 16 ◽  
Author(s):  
Mamdouh R. Rezk ◽  
Mina Wadie ◽  
Soheir A. Weshahy ◽  
Mahmoud A. Tantawy
Keyword(s):  
Minor Component ◽  
Time Saving ◽  
Prostate Hyperplasia ◽  
Spectrophotometric Methods ◽  
Ratio Difference ◽  
Ich Guidelines ◽  
Mean Centering ◽  
Benign Prostate ◽  
Urological Diseases

Background: Alfuzosin is recently co-formulated with solifenacin for relieving two coincident urological diseases, namely; benign prostate hyperplasia and overactive bladder Objective: Herein, green, simple and rapid spectrophotometric methods were firstly developed for simultaneous determination of the two cited drugs in their co-formulated pharmaceutical capsule Methods: Alfuzosin, which is the major component in the dosage form, was directly assayed at its extended wavelength at 330.0 nm. The challenging spectrum of the minor component, solifenacin, was resolved by five spectrophotometric methods, namely; dual wavelength (DW) at 210.0 & 230.0 nm, first derivative (1D) at 222.0 nm, ratio difference (RD) at 217.0 - 271.0 nm , derivative ratio (1DD) at 223.0 and mean centering of ratio spectra (MC) at 217.0 nm Results: The Proposed methods were successfully validated as per ICH guidelines. Alfuzosin showed linearity over the range of 4.0 - 70.0 μg/mL, while that of solifenacin were 4.0 - 50.0 μg/mL for DW, 2.0 - 70.0 μg/mL for 1D and RD methods, 1.0 - 70.0 μg/mL for 1DD and 4.0 - 70.0 μg/mL for MC method. Statistical comparison with their official ones showed no noticeable differences. The methods showed good applicability for assaying drugs in their newly combination. Besides eco-scale, the greenness profile of the methods was assessed and compared with the reported spectrophotometric one via the newest metric tool; green analytical procedure index (GAPI). Conclusions: The proposed methods are superior in not only being smart, accurate, selective, robust and time-saving, but also in using distilled water as an eco-friendly and cheap solvent

scholarly journals A Toolbox for the Determination of Nitroaromatic Explosives in Marine Water, Sediment, and Biota Samples on Femtogram Levels by GC-MS/MS

Toxics ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 60
Author(s):  
Tobias Hartwig Bünning ◽  
Jennifer Susanne Strehse ◽  
Ann Christin Hollmann ◽  
Tom Bötticher ◽  
Edmund Maser
Keyword(s):  
Sample Preparation ◽  
Freeze Drying ◽  
Solid Phase ◽  
Phase Extraction ◽  
Direct Extraction ◽  
Time Saving ◽  
Marine Samples ◽  
Volume Method ◽  
Biota Samples

To determine the amount of the explosives 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and its metabolites in marine samples, a toolbox of methods was developed to enhance sample preparation and analysis of various types of marine samples, such as water, sediment, and different kinds of biota. To achieve this, established methods were adapted, improved, and combined. As a result, if explosive concentrations in sediment or mussel samples are greater than 10 ng per g, direct extraction allows for time-saving sample preparation; if concentrations are below 10 ng per g, techniques such as freeze-drying, ultrasonic, and solid-phase extraction can help to detect even picogram amounts. Two different GC-MS/MS methods were developed to enable the detection of these explosives in femtogram per microliter. With a splitless injector, limits of detection (LODs) between 77 and 333 fg/µL could be achieved in only 6.25 min. With the 5 µL programmable temperature vaporization—large volume method (PTV-LVI), LODs between 8 and 47 fg/µL could be achieved in less than 7 min. The detection limits achieved by these methods are among the lowest published to date. Their reliability has been tested and confirmed by measuring large and diverse sample sets.

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