What precautions would you take in competent cells formation and transformation of competent cells with recombinant...
What precautions would you take in competent cells formation and transformation of competent cells with recombinant plasmid DNA
Homework Answers
Precautions taken during transformation-
- The transformation is effective when <10ng of DNA is used. PUC19 DNA suitable as control.
- Supercoiled DNA is most efficient for transformation compared to linear or single stranded DNA
- In electroporation, presence of salts might lower transformation efficiency. Limit the
- volume of plasmid DNA to 1L per transformation
- Column-purified DNA might be good choice
- Ligation mixtures inhibit transformation. ligases must be heat
- inactivated before the mixture is added to the cells.
Heat shock -
- 42°C for 45 seconds for PCR tubes or thin-walled tubes
- . 37°C for 60 seconds for microfuge tubes or thick-walled tubes
- General set up: 37°C for 60 seconds
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1. Fill in the table above with what you observe on your plates. 2. Bacterial transformation...
1. Fill in the table above with what you observe on your
plates.
2. Bacterial transformation occurred on which agar plate (s)?
What evidence do you have that the bacteria were transformed
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3. Which plates have glowing growth? Explain what causes
bacteria to glow.
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Please classify each statement as describing transformation, conjugation, or transduction in bacteria. Bacteria can acquire plasmids from outside the cell. A bacterium that contains an F plasmid connects to a recipient bacterium that lacks an F plasmid with an appendage called a pilus, through which the plasmid is transferred. Some bacterial DNA fragments may be included when new phage particles are assembled. A cell can be treated to make it competent to take up DNA from its environment. When a...
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4. You have prepared a batch of competent E coli cells and found its transformation efficiency is 106 per μg plasmid DNA. A professional scientists prepared competent E. coli cells having a transformation efficiency of about 108 cells/ g plasmid DNA Calculate % score of your E. coli transformation efficiency (Τ.Ε.).
1. Fill in the table above with what you observe on your
plates.
2. Bacterial transformation occurred on which agar plate (s)?
What evidence do you have that the bacteria were transformed
here?
3. Which plates have glowing growth? Explain what causes
bacteria to glow.
II. Transformation of E. coli with Plasmid DNA (PGLO) 1. Three LB (Luria Broth) agar plates are obtained. The plates contain: • Plate A: LB-Agar/Ampicillin/Arabinose • Plate B: LB-Agar/Ampicillin • Plate C: LB-Agar 2. Three...
RECOMBINANT DNA: PLASMID VECTOR engineering is the direct manipulation of an organism's DNA using nology. To begin the recombinant DNA process, scientists must first ide at codes for the production of the protein they want to manufacture. One is to go backwards from the amino acid sequence of the desired protein to ide sequence of the gene. After scientists have identified the gene, they m it. Restriction enzymes or endonucleases from bacterial cells are key in th ia produce restriction...
Please classify each statement as describing transformation, conjugation, or transduction in bacteria. Bacteria can acquire plasmids from outside the cell. A bacterium that contains an F plasmid connects to a recipient bacterium that lacks an F plasmid with an appendage called a pilus, through which the plasmid is transferred. Some bacterial DNA fragments may be included when new phage particles are assembled. A cell can be treated to make it competent to take up DNA from its environment. When a...
2) With reference to production of recombinant DNA answer the following questions: i) what is "plasmid" and how is it isolated from a cell (1 point); ii) what is "insert" (1/2 point); iii) name the enzyme used for cleaving and generating sticky ends on the plasmid and insert (1 point); iv) name the enzyme used for joining sticky ends of plasmid and insert to produce a molecule of recombinant DNA (1 point); v) name the most common microbial host cell...
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