Preparation of homemade molecular weight markers

Project Title

Preparation of homemade molecular weight markers

Project Area of Specialization Biomedical EngineeringProject Summary

Gel electrophoresis is one of the routine procedures carried out in molecular biology labs for the purpose of visualizing the DNA bands. For assessing the parameters of DNA bands which include size, quality, and quantity, the molecular weight markers are used. These molecular weight markers comprise a pool of DNA fragments whose sizes are known. This study is the applied research that highlights an economical way of preparing DNA ladders that will be eligible for a common researcher to apply it in the laboratories. During this project, pPSU plasmids will be used as a source for the preparation of DNA molecular weight markers. For this purpose, initially, we will be going to isolate pPSU1 and pPSU2 plasmids from commercially available competent strains of pPSU plasmids transformed E. coli. The plasmids will then undergo digestion with the restriction enzyme EcoRV and Pst1 separately and together to generate fragments of different sizes, which will be used to synthesize homemade molecular weight markers of 1Kb and 100 base pairs. These homemade molecular weight markers will not only be useful in the determination of nucleic acid size however it will also be cost-effective for the research community.
 

Project Objectives

The molecular weight markers are the most common reagents used in molecular biology laboratories; this signifies it's usage in extended amounts. The commercial molecular weight markers cost ranges from $200-$500 to run 1000 lanes while the 1KB and 100 base pairs ladders prepared through home-made plasmid extraction and digestion protocol which is also sufficient to run 1000 lanes cost only less than $10. To get a DNA ladder in flexible prices we will prepare the 1KB and 100 base pairs ladder in our laboratory.

The objective of this study is to prepare a home-made molecular weight marker in order:

a)         To predict the size of unknown DNA fragments using the pair of digested pPSU1 and pPSU2 plasmids having known molecular weight fragments.

b)         To synthesize the high-quality molecular weight marker in cost-effective prices in laboratories

Project Implementation Method

The Method of this project will be consisting the following steps:

 In the First step, E. coli strain (DH5?) bearing pPSU1 & pPSU2 will be grown in Luria Bertani (LB media), which will be prepared using yeast, tryptone, and sodium chloride.

For the culturing of E. coli strain (DH5?), 1mM ampicillin will be added in LB media to enhance selective growth of E. coli cells because pPSU1 & pPSU2 plasmids contain Ampicillin resistant gene. E. coli cells will be inoculated in LB media and will be kept overnight on shaking incubator at 37 o C.

Second step will be the extraction of the Plasmid DNA using the alkaline lysis method. This method comprises of three solutions consisting of different ingredients which will enable the extraction of plasmid. Isopropanol will be added in the latter steps to precipitate Plasmid DNA. To elute the Plasmid DNA, an elution buffer will be used.

Third step will involve the visualization of the plasmid DNA through gel electrophoresis and visualizing will be done using the U.V illuminator. 

 In the fourth step digestion of plasmid DNA will be done using two restriction endonucleases which are EcoRV and Pst1.

The Last step is about visualizing the digested Plasmid DNA through gel electrophoresis and visualizing through a U.V illuminator.

Benefits of the Project

A DNA ladder or molecular weight markers is a pool of different DNA fragments having different sizes that are known. Determining the sizes of unknown DNA fragments on gel electrophoresis is one of the routine procedures held in molecular biology labs which is done by using DNA ladders, moreover, it also helps in predicting the properties of unknown DNA samples. They are used as an estimation to predict the size of unknown DNA fragments in a variety of procedures such as northern and southern blotting, PCR amplification, in fact in every DNA related procedure. DNA ladders have also been used in other numerous fields as well such as biotechnology, medicine, and agriculture therefore its extended usage justifies its demand in the market.

DNA ladders being prepared commercially as well as in laboratories have certain restrictions. In laboratories, they are prepared through restriction digestion of lambda phage DNA or plasmids. However, looking for a cheaper source of lambda phage DNA is another thought-provoking domain. The DNA ladders developed commercially are of high quality as well as of high prices, the high prices restrict its usage because of limited capital availability everywhere

To eradicate these limitations associated with commercially available DNA ladders, we decide to prepare homemade DNA ladders in cheaper rates in our laboratory, using restriction digestion of plasmids pPSU1 and pPSU2 with EcoRV to produce 1Kb and 100 base pairs ladders. In comparison to other methodologies for preparing the DNA ladder, this one will be the simplest and flexible way of preparing DNA ladders in terms of being economical and easily accessible. Moreover, this method will also facilitate researchers to work in common laboratories.

Technical Details of Final Deliverable

The proposed project is based on the molecular biology approach.

During this project pPSU plasmids will be used as a source for the preparation of DNA molecular weight markers. For this purpose, initially, we will isolate pPSU1 and pPsu2 plasmids from commercially available competent strains of pPSU plasmids transformed E. coli. These pPSU plasmids after isolation will then undergo digestion with two of the restriction enzymes EcoRV and Pst1 separately and together After digestion fragment of different sizes will be obtained from both plasmids that can be used to prepare 1kb and 100 base pair molecules weight markers which will be applicable to run across 1000 gels.

Final Deliverable of the Project Hardware SystemCore Industry EducationOther Industries Medical Core Technology OthersOther Technologies OthersSustainable Development Goals Responsible Consumption and ProductionRequired Resources

Item Name	Type	No. of Units	Per Unit Cost (in Rs)	Total (in Rs)
			Total in (Rs)	40752
Tips 10 microlitre	Equipment	1	3500	3500
Tips 100 microlitre	Equipment	1	3500	3500
Tips 1000 microlitre	Equipment	1	1800	1800
Tryptone	Equipment	1	12500	12500
yeast extract	Equipment	1	7500	7500
EcoRV	Equipment	1	5928	5928
PstI	Equipment	1	6024	6024