Team:IISc-Bangalore/Protocols

  1. Transformation
  2. Plasmid isolation
  3. PCR
  4. DNA purification
  5. Restriction digestion
  6. Ligation
  7. SEM assay
  8. DLS assay
  9. Spectrophotometry assay
  10. Reagents

Transformation

Preparation of competent cells - TSS method

For E. coli cells to take up a foreign plasmid, they have to first be made competent - their cell walls must be weakened and made permeable to incoming DNA. This is traditionally done using divalent cations, which mask the negative charge of the phospholipid membrane, allowing the negatively-charged DNA to approach the cell without significant repulsion. The Transformation and Storage Solution (TSS) contains Mg++.

Day 1

Step Description Rationale
1 Prepare a primary inoculum of E. coli in 5 mL LB in a test tube. -
2 Incubate overnight at 37°C, 170 rpm. -

Day 2

Step Description Comments
1 Inoculate 1 mL overnight culture in 100 mL LB medium (1% inoculum) This protocol yields one aliquot of competent cells per mL of culture. Depending on how many aliquots of competent cells you wish to make, you can vary the volume of LB medium used.
2 Incubate at 37°C, 170 rpm until the OD reaches 0.4 (~1 h 45 min for E. coli DH5α cells) This is the early exponential phase; cells are physiologically ideal for the preparation of competent cells.
From this point, cells should always be placed in the cold (below 4°C), all buffers should be ice-cold, and all plasticware/glassware should be pre-chilled.
3 Place the culture at 4°C for 45 min -
4 Spin down the culture at 10000 rpm, 10 min, 4°C -
5 Resuspend the cell pellet in 1 mL ice-cold TSS buffer -
6 Spin down the culture at 10000 rpm, 10 min, 4°C -
7 Make 50-100 uL aliquots in chilled microfuge tubes, snap-freeze in liquid nitrogen and store at -80°C for long-term storage -

Heat-shock transformation

Plasmid isolation

Miniprep

Midiprep

PCR

Simple PCR

Overhang PCR

Colony PCR

DNA purification

Chloroform extraction

Gel purification

Restriction digestion

Single digestion

Double digestion

Overnight digestion

Ligation

Simple ligation

Sequential ligation

Multi-ligation

Scanning Electron Microscopy (SEM) assay

Dynamic Light Scattering (DLS) assay

Spectrophotometry assay

Stocks

LB medium

TSS buffer

Reagent Volume Final concentration
2x LB medium 0.5 mL 5% (v/v)
PEG 3350 0.5 mL 5% (v/v)
DMSO 0.5 mL 5% (v/v)
2M MgCl2 0.5 mL 0.1 MgCl2
MilliQ water 8 mL Up to 10 mL

Chloramphenicol stock solution

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