Table 2 Restriction endonuclease digest of genomic DNA Reaction components gDNA Hpall OR Mspl . Restriction endonuclease buffer Water Details 200 ng/μl Mspl: 20 units/μl Hpall: 10 units/μl Provided as 10x stock Add to make final reaction volume 20μl Amount in final reaction 1ug 20 units 1x in final reaction Add to make final reaction volume 20μl Volume in final reaction (μl) DNA digestion 1hr, 37°C Remember that you need to set up one tube for Mspl and 1 tube for Hpall, see Figure 7
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
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Reserve for analysis
using agarose gel
electrophoresis (250ng)
- 100% + B
Elective Ideas
Activity 1: Digestion of Genomic DNA with Hpall or Mspl
Water
1μg genomic DNA
20 units of Mspl per ug DNA
Reaction volume 20μl
Hpall OR Mspl*
Digest 90 mins 37°C
Take 500ng of digested DNA
Sequencing adaptor final concentration
0.5 or 1μM
Ligation reaction 10 mins
Reaction volume 20μl
Take 50ng, PCR reaction
Column-based purification of PCR products
Nanodrop quantification of products
Analyse up to 200ng
using agarose gel
electrophoresis
Reaction components
gDNA
Table 2 Restriction endonuclease digest of genomic DNA
Restriction endonuclease
buffer
b
1μg genomic DNA
20 units of Hpall per pg DNA
Reaction volume 20μl
Digest 90 mins 37°C
Take 500ng of digested DNA
Sequencing adaptor final concentration
0.5 or 1μM
Ligation reaction 10 mins
Reaction volume 20μl
Figure 7. Diagram showing the setup of digestion of human DNA with Mspl or Hpall. These
reactions are set up in 0.2ml (200μl) tubes. The gDNA is extracted from human circulating white
blood cells and is provided at a concentration of 0.2mg/ml (200ng/ul).
Take 100ng, PCR reaction
Column-based purification of PCR products
Nanodrop quantification of products
Details
200 ng/μl
Mspl: 20 units/μl
Hpall: 10 units/μl
Provided as 10x stock
Add to make final
reaction volume 20μl
SAMSUNG
Analyse up to 200ng
using agarose gel
electrophoresis
H
Amount in final
reaction
1ug
20 units
1x in final reaction
Add to make final
reaction volume
20μl
Reserve for analysis
using agarose gel
electrophoresis
W
DNA digestion
1hr, 37°C
* Remember that you need to set up one tube for Mspl and 1 tube for Hpall, see Figure 7
Volume in final
reaction (μl)
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