To Joyce: These are brief protocols including competent cells preparation, DNA transformation and DNA purification. These protocols are standard ones. Although some of these protocols are different from what you have learned, you could still write these in your proposal.
Chemicals: LB medium, TSS solution, TE, LB/Amp agar plates,
Bacterial strain: E. coli TOP10F'
Equipment: Pipetman, microcentrifuge, spectrophotometer, orbital shaker
incubator, water bath, incubator, L-shape glass rod.
I. Preparation of Competent Cells
1. Inoculate one single colony of bacterial host cells into 5 mL of LB
medium and incubate at 37 with shaking at 220 rpm for 2.5 to 3 hr until the culture becomes cloudy (OD590nm=0.4).
2. Transfer 1.5 mL of LB culture into a microcentrifuge tube and centrifuge at 6,000 rpm for 30 sec at r.t.
3. Decant the supernatant and wipe the wall of the microcentrifuge tube with kimwipe.
4. Resuspend the host cells in 100 ul of TSS solution.TSS solution (Transformation and Storage Solution for chemical transformation)
TSS 100 mL 10 mL
85% LB medium 80 mL 8 mL
10% PEG (polyethylene glycol wt/vol: MW 8,000) 10 g 1 g
5% DMSO (vol/vol) 5 mL 0.5 mL
50 mM MgCl2 (pH 6.5) 1 g 0.1 g
H2O 10 mL 1 mL
*Autoclave or filter to sterilize the solution, store at 4 for up to two weeks.
II. Transformation
1. Preheat the water bath to 42*C.
2. Add 5 ul of DNA to 100 ul of competent cells, swirl the tubes gently several times to mix.
The volume of the DNA solution should not exceed 5% of the volume of the competent cells for optimal results.
3. Add 5 ul of ddH2O or TE to 100 ul of cells as the negative control.
4. Add 1 ng of supercoiled plasmid vector to 100 ul of cells as the positive control.
5. Place all three tubes on ice for 20 min.
6. Transfer tubes to a floater in a 42 *C circulating water bath. Leave the tubes in the water bath for exactly 1 min. Do not shake the tubes.
7. Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 1-2 min.
8. Add 1 mL of LB medium (w/o antibiotics) to each tube. Mix gently.
9. Incubate the tubes at 37*C with shaking at 220 rpm for 45 min to
allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
10. Centrifuge at 6,000 rpm for 30 sec at r.t. and remove 800 ul of supernatant.
11. Resuspend the cells in the remaining solution and transfer 100 ul of cells onto an LB agar plate containing 50 ug/mL ampicillin.
12. Gently spread the transformed cells over the surface of the agar plate with a sterile L-shape glass rod. Rinse the glass rod with absolute EtOH and sterile it by flame.
13. Leave the plates at r.t. until the liquid has been absorbed.
14. Invert the plates and incubate at 37 *C for 12-16 hr..
15. Count the numbers of single colonies obtained from the plate.
Isolate plasmid DNA
1. Centrifuge 10-100ml cells at 10000xg for 10 mins at 4℃.
2. Resuspend the cell in 3ml of resuspend solution.
3. Add 3ml Cell lysis solution and mix by inverting 4times.(don't extend beyond 3-5 mins)
4. Add 3ml of neutralization solution and mix by inverting the tube 4 times.
5. Centrifuge the lysate at 14000xg for 15 mins at 4℃.
6. Decant the s/n to a new tube and avoid the white precipitate.
7. 10ml resuspend resin to s/n.
8. Transfer resin/ DNA mix into Midicolumn.
9. Add 15ml wash solution to the Midicolumn and centrifugation.(2 times)
10.Add 10ml 70% ethanol and centrifuge at 4000rpm for 10mins.
11.Place the washed column to a new tube and add 0.7ml preheated (60-65℃) water. Centrifuge at 3000 and 4000rpm for 10mins.
Pipete the DNA to 1.5ml tube.
12. Analyzing the DNA on an agarose gel electrophoresis.