What is the copy number of pBR322?
For example, pBR322 is a medium copy number plasmid (~20 copies/cell) from which several high copy number cloning vectors (>100 copies/cell) have been derived by mutagenesis, such as the well known pUC series.
How do you determine a genes copy number?
To measure DNA copy number, the amplicon should be located either within an exon or intron with sequences unique to that gene. A control gene with two copies should also be included. A master mix containing all of the components is prepared and distributed in 96 or 384-well plate.
How is the copy number of plasmid vector?
The recombinant DNA can multiply as many times as the copy number of the vector plasmid thereby determining the yield of recombinant protein. So, higher the copy number of plasmid vector, higher the copy number of gene and consequently, protein coded by the gene is produced in high amount.
Is my plasmid high or low copy number?
A way to determine experimentally if the copy number of your plasmid is high or low is to perform a miniprep. A high-copy plasmid should yield between 3-5 ug DNA per 1 ml LB culture, while a low-copy plasmid will yield between 0.2-1 ug DNA per ml of LB culture.
How is plasmid copy number regulation?
Bacterial plasmids maintain their number of copies by negative regulatory systems that adjust the rate of replication per plasmid copy in response to fluctuations in the copy number.
What is meant by copy number?
Definition of copy number : a numeral placed on a book to distinguish it from other copies of the same title.
Is there any relation between plasmid size and copy number?
Plasmids vary widely in copy number depending on three main factors: 1) The ori and its constituents – (e.g. ColE1 RNA I and RNA II). 2) The size of the plasmid and its associated insert (bigger inserts and plasmids may be replicated at a lower number as they represent a great metabolic burden for the cell).
How many droplets are in pBR322 plasmid copy number?
Quantification of pBR322 plasmid copy number with a single colour, multiplex, digital droplet PCR. (A) Column represents a single well of ~ 20,000 droplets containing E. coli DH5α total DNA template with multiplexed bla and dxs.
How do you find the copy number of a single-copy plasmid?
Absolute and relative quantification methods were used to quantify the PCN [ 19, 20, 22 ]. Both, bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively. Therefore, the plasmid copy number can be determined as the copy ratio of bla to dxs: PCN = [copy number of bla ]/ [copy number of dxs ].
What is the size of pBR322?
1. pBR322 Introduction: pBR322 is a purpose built plasmid vector and was one of the first widely used cloning vector. It has a relatively small size of 4,363 bp. This is important because transformation efficiency is inversely proportional to size and above 10 kbp is very low.
What is the function of pbbr322?
pBR322 is an E. coli plasmid cloning vector containing the origin of replication from pMB1 (a plasmid in the ColE1 com-patibility group; 1–3). The rop gene product, which regulates plasmid replication by stabilizing the interaction between RNAI and RNAII transcripts, maintains the copy number at about 20 per cell.