2014 IMSD LAB Manual Chapter 2
Week2 Schedule
6/30 Transformation/plasmids 2.1 Plasmid transformation
7/1 DNA structure-function 2.2 Colony selection
7/1 DNA replication 2.3 Plasmid Miniprep
7/3 Restriction Enzymes 2.4 Restriction digestion
2.1 Transformation
Bacterial Transformation
Transformation Competent Cells
Transformation is the process of introducing nucleic acid, especially of plasmid DNA into bacteria. Many species of bacteria can naturally take up nucleic acid whenever nutrients or oxygen levels are low and the cells need to take in large molecules for food. Bacteria can also be rendered transformation competent by artificial means. When bacteria are in a state of competency, their cell membranes are permeable to large molecules such as DNA. Competency can be induced in E.coli by altering the cell membrane with divalent cations, such as Ca++ and Mg++, together with rapid changes of the temperature between hot and cold. The Ca++ treated cells are called Calcium competent cells. Another way to induce competency is by an electric shock in a process called electroporation that also disrupts the membrane structure. Due to the altered cell membrane, the competent cells are very fragile and need to be handled carefully.
Transformation is not an efficient process. Typically, only one in every 10,000 cells can effectively pick up the DNA. Transformation efficiency is defined as the number of bacteria transformed by 1 ug of DNA.
The number of bacteria can be determined by the number of colonies forming on an agar plate……… colony forming unit (CFU), assuming that each colony is the progeny from a single bacterium.
DNA carriers
When DNA plasmids are used to carry foreign DNA genes, they are called plasmid vectors. An essential function of the plasmid is to afford identification of the transformed cells from a background of untransformed cells. Most plasmid vectors carry the antibiotic resistant genes, such as G418 resistant (KanR) and Ampicillin Resistant (AmpR). Cells transformed by a vector with AmpR can survive in the presence of carbenicillin, while the untransformed cells are eliminated. This process is referred to as antibiotic selection.
Bacteria culture
Anti-biotic selective pressure should be maintained at all times, because prolonged culture can accumulate cells without the plasmid through two mechanisms:
1. resistant enzyme leakage into the culture medium from resistant cells
2. uneven distribution of plasmids in daughter cells; cells with fewer plasmids grow faster.
Protocol
A. Preparing Antibiotic-containing Agar Plates
1. Add 20uL of stock Carbenicillin (100ug/uL) into 80uL sterile H2O….. 2000ug total.
2. Add the solution to an agar plate and spread it on the surface.
3. Let the antibiotic solution soak into the agar at room temp.
4. The agar is 20mL per plate, antibiotic concentration is at l00ug per mL.
B. Transformation
1. Thaw 50 uL of frozen CaCl2 treated competent cells on ice.
2. Add 5uL of plasmid DNA to 50 uL of thawed cells and stir gently.
3. Incubate all tubes on ice for 40 minutes.
4. Incubate all tubes at 42C for 40 seconds……… Heat shocking.
5. Put the tubes back on ice for a few minutes.
6. Add 100 uL of SOC media to each; total volume about 150 uL in each tube.
7. Incubate at 37C for 40 minutes for the cells to recover and express the KanR gene.
8. Pour about 15 uL of the transformed cells onto one agar plate, label as 1X.
9. Add the remaining cells to a second agar plate and label as 10X.
10. Spread the cells evenly over the agar surfaces.
11. Invert the plates, label and incubate at 37C overnight.