Research Methods 2.2 Preparing Competent Cells

why are competent cells prepared

to ligate PCR product into a plasmid vector in order to amplify it in bacteria

describe the current step of the research including what is to be done next

currently the amplicon from the PCR product is present, and using bacteria furtehr amplification and getting single colonies is possible, however, bacterial cells need to be modified and made competent so that they make take up foreign DNA (plasmid)

what can bacteria be used to propagate

plasmid by growing large amounts of the same plasmid as a clone

competent cells

bacterial cells that are capable of taking in foreign DNA

transformation

introduces the plasmid into living bacterial cells so that it can be replicated, to conduct transformation a competent cell is taken and plasmid is introduced into it so it can take up foreign DNA. it is the process of introducing foreign DNA to a competent bacterial cell.

transfection

foreign DNA to a eukaryotic cell using lipids

electroporation

foreign DNA to a eukaryotic cell using electric shock

infection

foreign DNA to a eukaryotic cell using a virus

methods for transformation

heat shock transformation and electroporation

what do both methods of transformation require

competent cells which as bacterial cells that can take up DNA, and in this experiment E.coli is used because a small number of cells may be able to take up DNA

how can you increase the number of competent cells in a bacterial culture using heat shock

the bacteria must be washed to remove the growth medium and then resuspended in CaCl2 solution

how can you increase the number of competent cells in a bacterial culture using electroporation

cells must be washed repeatedly in a chilled buffer and resuspended in a chilled sterile solution that has very low ionic strength

what allows successful transformation in both heat shock and electroporation

cells must in solution and at a high concentration, cells must be kept cold at all times prior to transformation because they are extremely fragile at this stage and the cold keeps them inert. if the cells are warmed up they will start to die. they must also be handled gently, pipetted slowly, and never vortexed.

why does the cold temperature help in competency

the cold hardens the cell membranes of the bacteria and the heat shock rapidly melts or breaks the membranes which allows DNA inside the cells

significance of CaCl2

keeps the DNA in close proximity to the bacterial cell surface so that once the cells are heat shocked it is already close enough to get inside the cell, it helps DNA to bind to membrane for convenient entry

what must bacteria be to make competent cells

actively growing and healthy

what is important to use for competent cells

a fresh starter culture (less than 24 hours since inoculation) for the transformation or transformation efficiency will be severly reduced

what is c growth medium

a nutrient broth that helps the bacteria enter growth phase efficiently

what does transformation buffer have to assist

CaCl2 and DMSO that assist the plasmid DNA to pass through the lipid cell membrane, transformation efficiency becomes seriously compromised if the cells warm up

where is bacteria located in the experiment

the pellet