Miniprep method LiCl
- spin bacteria 30 sec max speed
- aspirate supernatant
- resuspend pellet in 800 µl TELT and 10 µl lysozyme
- pierce cap with needle, boil 3-4 minutes
- leave in ice a few minutes
- spin max speed 4ºC 10-15 minutes
- transfer supernatant in new tubes with 550 µl isopropanol, mix
- spin max speed 10 minutes
- apirate supernatant, add 150 µl 70% Ethanol, vortex
- spin max speed 2-5 minutes
- aspirate ethanol, resuspend DNA with TE or water + RNAseA
TELT buffer: 50 mM Tris-Hcl pH8.0
62.5 mM EDTA
2.5 M LiCl (MW = 42.39), 106 gr per liter
0.4% Triton X100
Lysozyme 100 mg/ml

Electrocompetent Cells
NOTE: cells to be kept on ice at all times
all solutions and rotors are to be cooled before use
prechill sterile water and 10% glycerol in cold-room
grow cells until they are in midlog phase growth
~120 50ul aliquots should be made from 500ml culture
1. grow 5ml culture of E.Coli O/N
2. seed 500ml broth w/ 5ml culture
3. grow at 37 °C 250rpm shaking ~4hrs until OD600 ~0.600
4. chill culture 15min on ice in cold room
5. divide culture between two 250m centrifuge bottles
6. pellet cells: 4500rpm 10min 4 °C
7. decant supernatant and add 50ml sterile water
8. resuspend cells by shaking ~100rpm 15min on ice in cold room
9. divide 50ml suspension between five 50ml conical centrifuge tubes
10. pellet cells: 5000rpm 20min 4 °C
11. decant supernatant and add 10ml sterile water
12. resuspend cells as in step #8
13. pool pairs of tubes (now have 6 tubes total)
14. pellet cells as in step #10
15. decant supernatant and add 10ml 10% glycerol
16. resuspend cells and pellet again as done previously
17. resuspend cellular pellets w/ 1ml 10% glycerol
18. ”snap” freeze in 50ul aliquots in 1.5ml eppendorf tubes in liquid Nitrogen bath
19. Store at -80 °C

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Last modified: October 10, 2005