Bacterial Transformation

Question 1

Why do we add DNA into cells?

Answer 1

to replicate/amplify DNA
to express protein
modify the genome

Question 2

What kind of cells can DNA be added to?

Answer 2

In transformation, bacteria and yeast.
In transfection, eukaryotic cells.
Electroporation can use any cell types.

Question 3

How is bacteria made competent?

Answer 3

They are made chemically competent with CaCl2

Question 4

What are eukaryotic cells cultured with?

Answer 4

lipid/DNA or CaPO4/DNA

Question 5

What does yeast use in transformation?

Answer 5

LiAc/PEG

Question 6

What is transformation?

Answer 6

the process by which genetic material is taken up and integrated into the genome of a cell, resulting in a heritable change in the genetic makeup of the organism. Electroporation, heat shock, or chemical treatment make cells more receptive to foreign DNA.

Question 7

What is transfection?

Answer 7

used to introduce foreign DNA or RNA into cells, typically for the purpose of studying gene function or manipulating gene expression. The term “transfection” is a combination of “transformation” (the process by which foreign DNA is integrated into the genome) and “infection” (the process by which viruses introduce their genetic material into host cells).

Question 8

What is electroporation?

Answer 8

Electroporation involves the use of an electrical pulse to create transient pores in the cell membrane, allowing DNA or RNA to enter the cell. Lipofection uses liposomes, which are lipid-based vesicles that can fuse with the cell membrane to deliver DNA or RNA into the cell. Viral transduction involves the use of a virus (such as a retrovirus or lentivirus) to deliver foreign DNA or RNA into the cell.

Question 9

Why is cloning done?

Answer 9

-Replicating genetic material, amplify PCR fragments
-Producing transgenic animals
-Studying stem cells
-Cloning can be used to preserve endangered species by creating genetically identical copies.

Question 10

How do you clone PCR fragments into a vector?

Answer 10

• The PCR fragment needs to be ligated into a plasmid vector
• PCR should leave blunt ends
• The plasmid vector should be cut to leave blunt ends
• Combine insert (PCR product), cut vector and DNA ligase
• Transform this reaction mixture into competent bacteria
• Plate on selective media, incorporating blue/white selection

Question 11

What happens after transformation?

Answer 11

to isolate the DNA from the transformed cells, using techniques such as plasmid extraction or genomic DNA isolation. This DNA can then be purified and analyzed to confirm that the desired genetic material has been successfully integrated into the host cell genome.

Question 12

What are the steps for the whole procedure of cloning?

Answer 12

1- ligation of the purified PCR fragment into a plasmid
2- transformation into competent bacteria
3- plating of bacteria in selective media and growing over-night
4- picking a colony and growing over-night in a liquid culture
5- performing miniprep protocol to extract and purify the plasmid+insert from the bacteria
6- setting up a digestion with EcoRI of the purified plasmids, to determine presence/absence of insert

Question 13

How do we check that the DNA we cloned is the one we want?

Answer 13

1. Restriction enzymes can be used to cut the DNA at specific sites, creating a pattern of fragments that can be visualized using gel electrophoresis. By comparing the pattern of fragments from the cloned DNA with the expected pattern based on the known DNA sequence, it is possible to confirm that the correct DNA fragment has been cloned.
2. DNA sequencing: DNA sequencing can be used to determine the exact sequence of the cloned DNA, allowing researchers to confirm that it matches the intended sequence. This method is highly accurate and can detect any mutations or errors that may have occurred during the cloning process.
3. Polymerase Chain Reaction (PCR): PCR can be used to amplify a specific DNA sequence from the cloned DNA, which can then be visualized using gel electrophoresis. By comparing the size of the amplified DNA fragment with the expected size based on the known DNA sequence, it is possible to confirm that the correct DNA fragment has been cloned.
4. Southern blotting: Southern blotting is a technique that can be used to detect specific DNA sequences in a sample. By hybridizing a labeled probe to the cloned DNA and visualizing the signal, it is possible to confirm that the correct DNA fragment has been cloned.

Question 14

What is restriction enzyme digestion?

Answer 14

Technique used to cut DNA at specific sites using enzymes known as restriction enzymes or restriction endonucleases. These enzymes recognize and cut DNA at specific sequences called restriction sites, which are usually palindromic sequences of 4 to 8 base pairs in length. When the DNA is cut by restriction enzymes, it produces fragments of different sizes that can be separated using gel electrophoresis, a technique that separates DNA fragments based on their size. By comparing the pattern of fragments produced by restriction enzyme digestion with the expected pattern based on the known DNA sequence, it is possible to confirm that the correct DNA fragment has been cloned or to detect mutations or variations in the DNA sequence.

Question 15

What are the steps involved in a plasmid mini prep protocol?

Answer 15

1. Pellet bacterial culture by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in Plasmid Resuspension Buffer
3. Add Plasmid Lysis Buffer
4. Add Plasmid Neutralization Buffer, gently invert tube until neutralized, and incubate
5. Centrifuge lysate
6. Carefully transfer supernatant to the spin column and centrifuge
7. Re-insert column in the collection tube and add Plasmid Wash Buffer
8. Add Plasmid Wash Buffer and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube
10. Add DNA elution buffer to centre of matrix then spin to elute DNA

Question 16

What is the difference between T7 and SP6 polymerases in cloning?

Answer 16

They recognise different promoter sequences. T7 polymerase recognizes a T7 promoter sequence, while SP6 polymerase recognizes an SP6 promoter sequence.

Question 17

What is T7 polymerase?

Answer 17

a bacteriophage RNA polymerase that recognizes a specific promoter sequence and produces RNA transcripts in a 5’ to 3’ direction. It is often used for in vitro transcription of DNA templates, as it can produce large quantities of RNA in a relatively short amount of time. In cloning, T7 polymerase is often used to generate RNA probes for gene expression analysis or for in vitro translation experiments.

Question 18

What is SP6 polymerase?

Answer 18

RNA polymerase that recognizes a different promoter sequence and also produces RNA transcripts in a 5’ to 3’ direction. Like T7 polymerase, SP6 polymerase is commonly used for in vitro transcription of DNA templates, and it can also be used to produce RNA probes for gene expression analysis.

Question 19

What is the T-A cloning technique?

Answer 19

T-A cloning is a molecular biology technique used to insert a specific DNA fragment into a plasmid vector. This technique is commonly used for gene cloning and recombinant DNA technology.

The T-A cloning technique involves adding a single nucleotide overhang to the 3’ end of a DNA fragment using Taq polymerase, an enzyme commonly used in PCR reactions. The overhang is usually an “A” nucleotide, which pairs with a “T” nucleotide on the complementary strand of the plasmid vector.

The plasmid vector used in T-A cloning is usually linearized with a restriction enzyme that creates a compatible overhang. The linearized vector and the PCR-amplified DNA fragment are then mixed together, and the “sticky ends” of the overhangs anneal, creating a stable recombinant DNA molecule. This molecule is then transformed into bacteria, where it can replicate and produce many copies of the recombinant plasmid.

The T-A cloning technique is useful because it is a quick and efficient method for inserting DNA fragments into plasmid vectors without the need for restriction enzymes to create specific restriction sites. The technique is also widely used for cloning PCR products and for creating libraries of DNA fragments for functional genomics studies.