DNA transduction into cells

Question 1

Why do we add DNA into cells?

Answer 1

• Replicate/amplify DNA
- Multiple further uses
• Express protein
• Modify the genome

Question 2

What are the different methods of adding DNA into different cell types?

Answer 2

• Transformation
-Bacteria
-Yeast
• Transfection
-Eukaryotic cells
• Transformation
-Bacteria made chemically competent (eg CaCl2)
-Yeast use LiAc/PEG
• Transfection
-Eukaryotic cells cultured with lipid/DNA or CaPO4/DNA
• Or electroporation can be used for all cell types
-High voltage for a very short period of time.

Question 3

how should we clone PCR fragments into a DNA vector?

Answer 3

• 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

except its not that quite accurate….because not all DNA polymerases are the same

Question 4

What is Taq DNA polymerase?

Answer 4

• Taq DNA polymerase leaves a single, overhanging A at the 3’ end of
the PCR product
• The overhanging T residues in the vector prevent re-circularization of
the empty vector
• The overhanging A residues in the PCR product are complementary to
the T and promote integration of the insert
• We use blue/white selection to increase the chances of selecting a
clone with an insert

Question 5

What happens after bacterial transformation?

Answer 5

• Pick a single colony
• Blue or white?
• Grow in the presence of antibiotic in a 2ml culture
• Take 1.5ml of bacterial culture and retain 0.5ml – IMPORTANT!
• Extract DNA from the 1.5ml culture (mini-prep)
• Check that the DNA is the one you want

Question 6

Briefly outline steps involved in cloning and purifying DNA fragments from PCR

Answer 6

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- checking the plasmid+insert product: restriction enzyme reaction and/or PCR