Why is pBluescript a good vector?
Agilent pBlueScript II Vectors are powerful cloning vectors for a range of research applications. Featuring an extensive polylinker with 21 unique restriction enzyme recognition sites, the vectors are suitable for a range of DNA sequencing and cloning processes.
Is pBluescript a phagemid?
The pBluescript vectors, e.g., pBluescript II KS/SK(+ / –) (3 kb), are phagemids derived from pUC19 and have essentially similar configurations as pGEM-Zf except that they have T7 and T3 promoters, respectively, flanking the MCS which is composed of 21 unique restriction sites (95).
Is pBluescript a cloning vector?
The pBluescript II phagemids (plasmids with a phage origin) are cloning vectors designed to simplify commonly used cloning and sequencing procedures, including the construction of nested deletions for DNA sequencing, generation of RNA transcripts in vitro and site-specific mutagenesis and gene mapping.
What is pBluescript SK?
Plasmid: pBluescript SK (-) Standard cloning vector (phagemid excised from lambda ZAP). The f1 (-) orientation allows rescue of antisense strand ssDNA.
How do you Subclone a gene?
There are four steps in the subcloning process: obtain the target fragment, connect enzyme vector and target fragment, transform in host cell, identify and screen. Choose an appropriate restriction enzyme to cleave the target fragment from the vector.
What is the size of Pbluescript?
Plasmid: pBluescript SK (+)
| Source/Vendor: | Stratagene |
|---|---|
| Plasmid Type: | Bacterial Expression |
| Cloning Method: | Restriction Enzyme |
| Size: | 2958 |
| 5′ Sequencing 1 Primer: | M13pUC-fwd |
What is insertional activation?
Definition. Insertional activation/inactivation refers to either activation of an endogenous gene which is located near an integrated transgene, or to disruption of a gene or other functional sequence by insertion of a transposable element.
Why subcloning is done?
Subcloning Strategies. Subcloning is a basic procedure in molecular biology for transfer of DNA inserts from one vector to another to gain functionality to study the sequence of interest.
What are Subclone cells?
Subcloning involves expanding a cryopreserved cell line, trypsinizing to isolate single cells, and spreading these diluted cells sparsely onto a tissue culture dish.
What is phagemid biotechnology?
A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. These vectors carry, in addition to the origin of plasmid replication, an origin of replication derived from bacteriophage.
What is insertional inactivation in genetic engineering?
Insertional inactivation is a technique used in recombinant DNA technology. In this procedure, a bacteria carrying recombinant plasmids or a fragment of foreign DNA is made to insert into a restriction site inside a gene to resist antibiotics, hence causing the gene to turn non-functional or in an inactivated state.
Why is insertional inactivation detected?
1 Answer. In insertional inactivation method, the presence of a chromogenic substrate gives blue coloured colonies in absence of an insert. Presence of an insert in the enzyme site do not produce colour. This is because insertional inactivation of the β-galactosidase has taken place due to the insert.
Why is the genetically modified plasmid added into a bacterial cell?
Plasmids are used in the techniques and research of genetic engineering and gene therapy by gene transfer to bacterial cells or to cells of superior organisms, whether other plants, animals, or other living organisms, to improve their resistance to diseases or to improve their growth rates or to improve any other …
What type of genes do plasmids carry?
Plasmids can contain the following types of genes: antibiotic resistance genes, transgenes and reporter genes. These types of plasmid genes may occur naturally or be engineered by scientists.
Is the pBluescript KS+ vector available from Addgene?
This vector is NOT available from Addgene. Standard cloning vector (phagemid excised from lambda ZAP). The f1 (+) orientation allows rescue of sense strand ssDNA. pBluescript SK (+) and pBluescript KS (+) differ by the orientation of the MCS.
Does pBluescript plasmid DNA retain its supercoiled state during DNA synthesis?
It is crucial to verify that the pBluescript plasmid DNA retains its supercoiled state during DNA synthesis. The analysis provides assurance that extensive DNA synthesis is mediated via D-loop migration and not through a DNA relaxation or rolling circle mechanism that could result from a contaminating topoisomerase or endonuclease activity]
How are PCR-amplified fragments inserted into pBluescript vectors?
Polymerase chain reaction (PCR)-amplified fragments from RNA, cloned DNA, or genomic DNA can be inserted directly into pBluescript vectors, bypassing the need to construct large libraries. If a new gene of unknown sequence is to be cloned, libraries will need to be generated for screening.
How many restriction sites are there in a pBluescript SK vector?
The pBluescript SK (−) phagemid vector contains a large polylinker (21 unique restriction sites) flanked by T3 and T7 phage RNA polymerase promoters. (e) pBluescript vectors.