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 µL aliquots in chilled microfuge tubes, snap-freeze in liquid nitrogen and store at -80°C for long-term storage -

Heat-shock transformation

Step Description Rationale
1 Thaw the competent cells on ice for 20 min -
2 Add 1-5 µL of plasmid DNA (10 pg - 100 ng) directly into the competent cells. -
3 Gently flick the microfuge tube to mix the cells and the DNA. -
4 Incubate on ice for 30 min. -
5 Heat-shock the cells at 42°C for ~45 s The optimal heat-shock duration varies from 30 s to 90 s depending on strain, batch, and method of preparation. In our experiments, 45 s gave us a high enough transformation efficiency to proceed.
6 Place the cells on ice for 5 min -
7 Add 950 µL SOC medium. -
8 Incubate at 37°C, 220 rpm for 1-2 h. The optimal incubation time varies depending on strain and antibiotic resistance marker used for selection. This step allows the few transformants to replicate and increase their plasmid number in the following generations.
9 Spread plate 100 µL of the culture on a selection plate. -
10 Spin down the remaining culture at 5000 rpm, 10 min, resuspend in ~100 µL medium, and spread plate on a selection plate. -
11 Incubate plates overnight at 37°C until transformant colonies are seen. -

Plasmid isolation - Miniprep

Day 1

Step Description Rationale
1 Inoculate one colony of the desired transformant in 5 mL of LB medium in a test tube. -
2 Incubate overnight at 37°C, 170 rpm. -

Day 2

Step Description Rationale
1 Spin down 1.5 mL of the overnight culture in a 1.5 mL microfuge tube at 5000 rpm, 10 min. Discard the supernatant. Repeat until all 5 mL has been pelleted. -
2 Resuspend the cell pellet in 200 µL alkaline lysis solution I using a micropipette. -
3 Keep the microfuge tube on ice for 5 min. -
4 Add 400 µL alkaline lysis solution II along the sides of the microfuge tube. -
5 Very gently invert the microfuge tube three times. -
6 Keep the microfuge tube on ice for 30 s. -
7 Add 300 µL alkaline lysis solution III. -
8 Gently invert the microfuge tube three times. -
9 Keep the microfuge tube on ice for 1 min. -
10 Spin at 13000 rpm for 10 min. -
11 Transfer the supernatant to a 2 mL microfuge tube. -
12 Add RNase A (5 µg/mL) to the supernatant, and incubate at 65°C for 30 min. -
13 Add an equal volume of chloroform to the microfuge tube. -
14 Spin at 13500 rpm, 10 min, 4°C -
15 Carefully transfer the upper aqueous layer to a new microfuge tube, avoiding white particles of protein at the water-chloroform interface. -
16 Repeat Steps 13, 14, and 15. -
17 Add an equal volume of ice-cold isopropanol. -
18 Incubate on ice for 30 min. -
19 Centrifuge at 14500 rpm, 30 min, 4°C. -
20 Discard supernatant and add 500 µL ice-cold 70% ethanol. -
21 Centrifuge at 14500 rpm, 30 min, 4°C. -
22 Discard supernatant, mark the position of the pellet, and leave the excess ethanol to dry. -
23 Resuspend the DNA pellet in 20 µL MilliQ. -

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
Filter sterilize the TSS buffer using a 0.22 µm filter.

Alkaline lysis solution I

Reagent Volume Final concentration
glucose - 50 mM
Tris-Cl (pH 8.0) - 25 mM
EDTA (pH 8.0) - 10 mM
MilliQ water - -
Autoclave the solution at 121°C, 15 psi, 15 min to sterilize

Alkaline lysis solution II

Prepare a fresh solution just before use.
Reagent Volume Final concentration
1 N NaOH 2 mL 0.2 N
10% SDS (w/v) 1 mL 1% (w/v)
MilliQ water 7 mL Up to 10 mL

Alkaline lysis solution III

Reagent Volume Final concentration
5 M KOAc 60 mL 3 M
glacial acetic acid 11.5 mL 11.5% (v/v)
MilliQ water 28.5 mL Up to 100 mL
Autoclave the solution at 121°C, 15 psi, 15 min to sterilize

Chloramphenicol stock solution

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