can you explain why we are hoping to obtain white colonies? (Please explain both why blue colonies lack a cloned insert, and why white colonies possess a cloned insert.)
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.
X-gal is used to help with what we call “blue/white screening.” X-gal is a substrate that the enzyme beta-galactosidase can convert to blue color. When transformation reactions are plated on LB plates with (amp and) X-gal, if an insert was not cloned into the SmaI-site of pUC19, the bacteria will be blue. If an insert was cloned into the SmaI-site of pUC19, the bacteria will be white. Based on the information in this question, and shown in Figure 1 below, can you explain why we are hoping to obtain white colonies? (Please explain both why blue colonies lack a cloned insert, and why white colonies possess a cloned insert.)
Fig. 1. Restriction map of pUC19 (2686 bp). Image obtained from the New England Biolabs e-catalog (https://www.neb.com/products/n3041-puc19 vector#Product%20Information_Properties%20&%20Usage). Note the presence of the lacZa gene, and note that the SmaI site (nt 412) into which (ideally) your insert was ligated is found within the lacZ ORF. Enzymes in the multiple cloning site (MCS) only cut the plasmid once; enzymes not part of the MCS that are listed in boldface also only cut the plasmid once.
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