Notebook
Day 1
Description of everything we do
Voici le titre du tableau
| Titre A1 |
Titre A2 |
Titre A3 |
Titre A4 |
| Titre B1 |
Valeur B2 |
Valeur B3 |
Valeur B4 |
02/06/2015
- Stock solution MgSO4 : 0,5M
Pour 500mL :
61,62g de MgSO4 (MM : 246,48)
qsp 500mL H2O stérile
- SOB Medium : 1 L
| Final [c] |
Composent |
Volume & Mass |
| 2% |
Bactotryptone |
20 g |
| 10 mM |
NaCl |
2 mL (5 M stock) |
| 2% |
Yeast Extract |
5 g |
| 2,5 mM |
KCl |
0,18 g |
| 10 mM |
MgCl2 |
5 mL (2 M stock) |
| 10 mM |
MgSO4 |
20 mL (0,5 M) |
| - |
H2O |
qsp 1L |
| pH media to 7 with NaOH, then autoclave |
- 2XTY Medium 500 mL
| Composent |
Volume & Mass |
| Bactotryptone |
8 g |
| NaCl |
2,5 g |
| Yeast Extract |
5 g |
| H2O |
qsp 500 mL |
03/06/2015
- Transformation Broth (TB): 1 L
| Final [c] |
Composent |
Volume & Mass |
| 10 mM |
Pipes |
3,02 g |
| 15 mM |
CaCl2 |
2,21 g |
| 250 mM |
KCl |
18,64 g |
Mix the Pipes, CaCl2, and KCl in 900 ml of millipore water. Add NaOH until pH is 6.7,
Don't worry, dust disappear after pH adjust.
Add MnCl2 (see below), stir, adjust volume to 1 L, then filter sterilize. Store at 4C.
| Final [c] |
Composent |
Volume & Mass |
| 55 mM |
MnCl2 |
10,89 g |
- Making DMSO Competent Cells : DAY ONE
Grow 12 ml overnight culture of favorite strain of E. coli in 2XTY at 37°C and 250 rpm (We use Denis’ DH5-alpha).
04/06/2015
- Making DMSO Competent Cells : DAY TWO
Inoculate 1 L SOB with 12 ml overnight culture.
Keep 5mL of SOB for initial OD.
Grow culture at 18C and 180 rpm (this temperature is really important as we see a 10-fold decrease in competency when we grow them at room temperature).
- Solution stock Chloramphenicol 34 mg.mL-1 (29,5 mL) :
Weigh out 1 g chloramphenicol and dissolve in 25 ml of 100% ethanol.
Make up volume to 29.5 ml with 100% ethanol.
Sterilize by filtration.
STORAGE : Aliquots of appropriate volume can be stored at -20 °C
- Solution stock Tetracycline 12,5 mg.mL-1 (80 mL) :
Weigh out 1 g tetracycline and dissolve in 75 ml of 1:1 vol/vol distilled water:ethanol.
Make up volume to 80 ml with 1:1 vol/vol distilled water:ethanol. (40mL ethanol + 40mL dH2O)
Sterilize by filtration.
STORAGE : Aliquots of appropriate volume should be wrapped in aluminium foil and stored at -20 °C
05/06/2015
-LB agar 250mL (x3)
| Composent |
Volume & Mass |
| Bactotryptone |
2,5 g |
| NaCl |
2,5 g |
| Yeast Extract |
1,25 g |
| Agar |
3 g |
| H2O |
qsp 250 mL |
| Adjust pH to 7.5 with NaOH and autoclave for 20 minutes |
| + Chloramphenicol (34 mg/ml in ethanol) |
75 µL / 250 mL medium {10 µg/ml (final)} |
| +Tetracycline (12,5 mg/ml in 50% ethanol) |
250 µL / 250 mL medium {12,5 µg/ml (final)} |
-LB liquid 500mL
| Composent |
Volume & Mass |
| Bactotryptone |
5 g |
| NaCl |
5 g |
| Yeast Extract |
2,5 g |
| H2O |
qsp 250 mL |
| Adjust pH to 7.5 with NaOH and autoclave for 20 minutes |
- Making DMSO Competent Cells : DAY THREE
Grow cells until A600 0.5-0.7
Blanck = SOB
A600 nm = 0,011
A600 nm = 0,008
=> So we incubate all week-end at 18°C and 120 rpm.