Mediated assembly of multiple dna fragments with short end homologies. This way, the resulting construct can be used for both microinjection and bacterial feeding. Likewise, the atrich genome of lactobacillus helveticus 65% at was cloned. Cloning of a plasmidencoded clindamycinresistance determinant.
Tvector pmd19 simple is supplied with a control insert 500 bp dna for positive control reactions. Based in cleveland, ohio, the gomedia team comes up with amazing stock footage for your graphic design projects. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy. The nucleotide sequence files available below are those used to produce the plasmid vector, viral and bacteriophage maps contained in new england biolabs catalog as well as the.
A sample of competent cells is made to take up puc19 with the two genes. A cloning vector is a small piece of dna that can be stably maintained in an organism, and into which a foreign dna fragment can be inserted for cloning purposes. Dna fragments into puc19 vector to study the ligation. Expression of target dna is enabled by the presence of a. We present arrays relation to pointers and consider the problems arising from their use. For example, telomeric repeats and other atrich fragments from.
Pcr to test primers and puc19 pcr product should be 2. It contains identical multiple cloning site mcs as puc18 vector except that it is arranged in opposite orientation. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. Cloning vector, puc19, was originally derived from parent plasmid pbr322. A versatile cloning plasmid for the expression of genes in mammalian cells. These changes are described in the full plasmid details pdf. Compared to a traditional approach of vector linearization using restriction digest, pcrmediated amplification of a linearized vector from a minute quantity of template plasmid dna followed by the elimination of the methylated template ensures a purer sample of the linearized vector with little circular vector dna. The vector length is 2686 bp and is isolated from e. I plan to insert these constructs into a puc19 vector with a.
When the insert dna fragment is recut from the vector, the pcr primers should be used to add a restriction site to the 5end for restriction enzyme digestion and subcloning. Highlights purified by chromatography using proprietary patented technology more than. This plasmid contains the multiple cloning site mcs from puc19 however it has been modified slightly to accommodate some restriction sites in our vector system. To ensure a pure culture, we must start with a single bacterium. The cloning vector may be dna taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. Thermo scientific puc19 vector is a small, high copy number, e.
Free vector samples by the talented design team of arsenalgomedia design studio. The difference is that pgem3z has two additional, short pieces of dna, each of which acts as the recognition site for attachment of an rna polymerase enzyme. These digested samples and the undigested puc19 and pbr322 were run on an agarose gel to determine sample. The molecule is a small doublestranded circle, 2686 base pairs in length, and has. As well, the 5 phosphate groups of digested puc19 were removed to decrease recircularization of the vector during the ligation reaction. Copycontrol fosmid library production kit with pcc1fos. For example, if 10 pg of puc19 yields 50 colonies when 50. Do not include ethidium bromide in the gel or running buffer. Although successful genomic sequencing with pucbased vectors has. These cloning vectors contain a multiple cloning site at the lacz region that enables recombinant plamids to be verified via bluewhite colony screening using agar plates containing iptg and xgal. The vector is manufactured by digesting supercoiled puc19 plasmid with the restriction enzymes psti and kpni, and tested for. Ambion puc 19 dna is digested to completion with sau3a i. Good advice t his chapter describes how vectors are copied and accessed through subscripting. Here you can download a freebies sample pack, so you get a taste of.
The digested puc19 samples were purified using a phenol extraction of dna sample and ethanol precipitation of dna 1. It contains the pmb1 origin of replication from pbr322, but it lacks the rop gene and carries a point mutation in the rnaii transcript g 2975 in pbr322 to a 8 in puc19. It has been constructed using the ampicillin resistance gene and the pmb1 origin of. The puc18 and puc19 plasmids enable successful cloning of large dna fragments larger than those cloned with a m mp18 rf phage vector. Another sample is made to take up another puc19 with a dna insert recombinant puc19. L of a typical pcr sample with an average insert length 400700 bp will give the proper ratio of 1. Plasmid sequence and snapgene enhanced annotations. These digested samples and the undigested puc19 and pbr322 were run on an agarose gel to determine sample concentrations figure 1.
One such benefit is the ability to produce large quantities of biological materials that. Most of col e1 was deleted except for an origin of replication and an antibiotic resistance gene. The effect of increasing plasmid size on transformation. Load 100 ng of the fosmid control dna in an adjacent gel lane as a control. The isolated puc19 and hindiiidigested lambda were then digested with ndei and gel extracted. We recommend using a dsrna expression vector such as pprt4p, pdonrdt7, or pl4440 section 2. It would be hard to get ligated a circular plasmid puc19 into the topo vector, so i would not be surprised that you had no colonies. Principle foreign dna inserted at the mcs interrupts the. A phagemid is a normal plasmid which derived from a coliphage and a plasmid.
Use text editor or plasmid mapping software to view sequence. The pcc2fos vector, a modification of the pcc1fos fig. Psfcmvpuc19 cmv puc19 mcs plasmid plasmid vector for. The puc19 linearized vector is provided as a part of infusion cloning kits, and is intended to serve as a control for infusion cloning. The molecule is a small doublestranded circle, 2686 base pairs in length. The pet vectors 4 vector characteristics and cloning strategy 4 ligationindependent cloning lic of. A target site for spontaneous insertion of is10 element in. The best controls for a colony pcr are the same ones used to verify if the colony pcr primers work in the first place.
If you are not planning to use bacterial feeding then any vector can be used to clone target gene cdnas. Then well yes, i think you set the positive control wrong. Use with snapgene software or the free viewer to visualize additional data and align other sequences. These controls are quick references you can use when you run your pcr products out on a.
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