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biosci

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2Â¥2019-06-16 11:15:32
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rabbitnxm

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2Â¥: Originally posted by biosci at 2019-06-16 11:15:32
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rabbitnxm

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4Â¥2019-08-09 22:36:05
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WilliamHJ

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Î÷ÃÅ´µÑ©170: ½ð±Ò+3, ¹ÄÀø»ØÌû½»Á÷ 2020-03-11 08:06:52
Preparation of the cells:
Day 1:
Inoculate 5 ml of G/L-SGM17B medium with L. lactis glycerol stock from -80 C and grow
at 30 C, without aeration, overnight
Day 2:
Inoculate 50 ml of G/L-SGM17B with pre-culture in a dilution of 1:100 and grow at 30 C,
without aeration, overnight
Day 3:
- Add 50 ml full-grown culture to 400 ml of G/L-SGM17B medium
- Grow the culture until OD600 is 0.2-0.3 (ca. 3 h)
- Spin down cells for 20 min at 6000 x g, 4 C
- Wash cells with 400 ml of 0.5 M sucrose, 10% glycerol (4 C) and spin down at
6000 x g (centrifugation speed may need to be increased during successive washing
steps)
- Resuspend the cells in 200 ml of 0.5 M sucrose, 10% glycerol, 50 mM EDTA (4 C),
keep the suspension on ice for 15 min and spin down
- Wash cells with 100 ml of 0.5 M sucrose, 10% glycerol (4 C) and spin down (6000 x g)
- Resuspend the cells in 4 ml of 0.5 M sucrose, 10% glycerol (4 C):
 Use 40 ¦Ìl per electroporation (keep on ice)
 Or store the cells in small portions at -80 C, let them defreeze on ice before use
Electroporation:
- Place 40 ¦Ìl cells in a pre-chilled electroporation cuvette with 1 ¦Ìl DNA (100-500 ng
vector DNA reconstituted in TE-, Tris-buffer, or distilled water; for transforming cells with
ligation product use 500-1000 ng DNA) and keep the cuvette on ice
- Use Biorad Genepulser with following adjustments:
2000 V
25 ¦ÌF
200 ¦¸
- Pulse (normal reading is 4.5-5 msec)
- Add 1 ml of G/L-M17B + 20 mM MgCl2 + 2 mM CaCl2
- Keep the cuvette for 5 min on ice and incubate 1-1.5 h at 30 C
- Plate 10 ¦Ìl, 100 ¦Ìl, 900 ¦Ìl on M17agar with glucose or lactose and antibiotics (depends on
plasmid)
- Incubate 1-2 days at 30 ¡ãC
Materials:
- G/L-SGM17B:
M17-Broth with: 0.5 M sucrose
2.5% glycine
0.5% glucose or 0.5% lactose (strain dependent)
Add the sucrose and glycine to the M17-B and sterilize 20 min 121 ¡ãC. Add sterile
glucose or lactose after cooling down.
- 0.5 M sucrose/ 10% glycerol
- 0.5 M sucrose/ 10% glycerol/ 0.05 M EDTA
L. lactis grows very slowly on G/L-SGM17B. Leaving out the sucrose is possible (Wells et
al., 1993) but can decrease the transformation efficiency.
The medium for cell recovery must contain MgCl2 and CaCl2.

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5Â¥2020-03-10 11:17:16
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