3) How can you use PCR to clone a 2000bp gene into a plasmid vector?

Solutions

Expert Solution

PCR based cloning is different from the traditional method in which gene of interest and vector both are amplified using polymerase chain reaction (PCR). Both the gen of interest and vector are ligated without the use of restriction enzymes. This process is advantageous than traditional method being rapid and can clone gene fragments that are not available in large amounts.

The following steps are involved in the PCR based cloning method:

1. Designing of primers for PCR based cloning

Primer design for pCR based cloning should consists of; Leader sequence, Restriction site and Hybridization sequence.

Leader sequences are 3-6 base pairs on the 5’ end of the primer

Restriction site (6-8 bp) depends on the individual doing an experiment. This should be determined precausiously as it should not cut within the gene of interest and must be present at desired location of the vector (multiple cloning site).

Hybridization region (18-21 bp) is the sequence of primer that binds to the gene sequence to be amplified.

2. PCR reaction is carried out to amplify the gene of interest using high fidelity taq polymerase for reducing mutation

3. Isolation of PCR product from the PCR reaction

4. Preparation of DNA digest and vector plasmid using restriction enzymes

5. Isolation of gene and plasmid vector by gel purification technique so as to have a desired gene size

6. Ligation of gene of interest into the vector

Typical DNA ligation is required to insert the gen of interest into the plasmid vector

7. Transformation of ligated plasmids into competent cells like DH5α. The number of colonies obtained is the indication of good transformation

8. Isolation of plasmids with gene of interest using overnight grown cultures. Bacterial colonies picked from the transformed plates will be grown and DNA purification is carried out on DNA using restriction enzymes that were used earlier. The product is run on agarose gel and provide you two bands, vector and insert.

gladiator answered 1 year ago

Next > < Previous

Related Solutions

You decide to clone a gene using a plasmid vector featuring ampicillin resistance and the lacZ...

You decide to clone a gene using a plasmid vector featuring ampicillin resistance and the lacZ gene. However, when you try to grow the E. coli onto a plate containing X-gal and ampicillin, you see only blue colonies. Explain what most likely went wrong during the cloning process.

How would you clone the Eukaryotic gene into the plasmid How would you transform bacteria with...

How would you clone the Eukaryotic gene into the plasmid How would you transform bacteria with that engineered plasmid How would you select for the bacteria that will take up the plasmid with integrated Eukaryotic gene

1) You wish to clone a gene into a bacterial plasmid for the production of the...

1) You wish to clone a gene into a bacterial plasmid for the production of the E protein to generate antibodies, use in vaccine production, and for biochemical analysis. A. What expression plasmid will you use and why? B. What primers to facilitate cloning into this expression vector? C. What essential features (DNA sequence elements) must the plasmid have to ensure: i. Plasmid maintenance/amplification in bacteria ii. Selection of cells transformed with your plasmid iii. Controlled/inducible expression of Gene E...

Describe in detail how to clone your “gene of interest” into a bacterial plasmid using EcoRI...

Describe in detail how to clone your “gene of interest” into a bacterial plasmid using EcoRI restriction enzyme and how to generate a large quantity of the DNA using competent E. coli. Why do you use competent bacteria for this?

Describe how you can use PCR to obtain a gene fragment X that can be cloned...

Describe how you can use PCR to obtain a gene fragment X that can be cloned into a plasmid vector for expression of the recombinant protein. Include in your description how the PCR reaction will be performed, how the amplicon will be analyzed, the use of restriction enzymes, how cloning will be achieved, how you will know that you have a recombinant containing the insert gene fragment X and how you will detect the recombinant protein.

Describe how you can use PCR to obtain a gene fragment X that can be cloned...

In order to clone a DNA gene, it is inserted into a larger vector. Why should...

In order to clone a DNA gene, it is inserted into a larger vector. Why should the vector used be larger?

Make a diagram to explain how a human gene can be cloned into a bacterial plasmid....

Make a diagram to explain how a human gene can be cloned into a bacterial plasmid. You should represent the events that need to occur in order to: Clone the insulin gene into a bacterial plasmid, Transform the plasmid into E. coli Include definitions or descriptions of the following terms/components: Restriction enzyme(s) Ligase Plasmid DNA Human mRNA (the mRNA for insulin) Reverse transcriptase Transformation E. coli marker genes (an antibiotic resistance gene) Cloning vector

Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector Foregin DNA = 720 Plasmid...

Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector Foregin DNA = 720 Plasmid DNA 2804 Post-Lab Questions 1. What is the expected size of the plasmid plus the cut foreign DNA? 2524 base pairs 2. What type of ends do the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning? 3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does...

You are inserting your gene of interest into the Lac Z gene in a plasmid also...

You are inserting your gene of interest into the Lac Z gene in a plasmid also containing a tetracycline resistant gene. You plate your transformed bacteria on media containing tetracyline and X-gal. Which of the following results would indicate a clone with recombinant plasmids? Select one: a. A blue colony on the tetracyline plates b. A clone which did not grow on the tetracycline plates c. A red colony on the tetracyline plates d. A white colony on the tetracyline...

Subjects