EPF-Lausanne/1 September 2009

Wet Lab

Results of yesterday transformation

Both plates grew (RFP test and CFP) We can see a couple of clones on each plate -> 2 clone son each plate picked in were put in 5ml LB + antibiotic, and put at 37°C (9h00)

Transformation

The 2 RO1 (gel & purified) that have ligated overnight at 4°C have been transformed in competent DH5 a. They were then plated on LB/agar + Amp plates. -> put in incubator at 37°C

Second test for the digested PCR Klenow for purity: Apparently no DNA in the last, and maybe not even in the first two.

Klenow protocol

New protocol used with no purification step, everything is prepared to go trough all reaction up to the end of the digestion, ready for the ligation without going through any step of purification. This will avoid losing DNA due to the small size of the TrpO (127bp).

Goal: we want 100ng/ul od DNA at the end
length of TrpO:127bp
molecular weight: 78,522 . 10^3 g/mol
→ 100ng/ul in 50ul → 6,367.10^-11 mol of TrpO a the end, that's to say 6,367.10^-11 of each primer

Trp Operon-Rev: 1,560.10^3M → 2.56 ul Trp Operon-Fwd: 1,560.10^3M → 2.56 ul

first make a dilution at 25ul in 500ul → 8ul in 492ul of MQ

1. Klenow
dNTPs final concentr = 1mM

3.64ul of NEB2

0.37ul of BSA 100x

2.56ul of each primer at 25uM

32.38ul of MQ

→ final: 36.4ul

Thermal cycler:

• 94°c for 5min
• 0.1°C/s to 74°C for 5 min
• 0.1°C/s to 37°C

Annealing temperature: 79.1°C (due to some website)

1.' 1ul Klenow + 1.6ul dNTPs = vol 39ul Incubate 1h30 at 37°C, then inactivate 20 min at 75°C : 0.1°C/s to 37°C

2. Digestion
Add 0.5 ul of each enzyme (EcoRI - HF / NheI) → vol: 40.0ul Incubate 2h at 37°C, then inactivate 20min at 80°C. Then cool down at 37°C (0.1°C/s)

3. Dephosphorylation
Add 5ul of antartic phosphatase buffer + 5ul of phosphatase enzyme → total volume: 50ul Incubate 2h at 37°C, then inactivate 10min at 65°C. Then cool down slowly (0.1°C/s)

1ul is needed to dephosphorylate 1-5mg of vector pUC19

Medium

New media were tried to find one where DH5a can grow but containing no TRP. All media were designed for 100ml.

• SC medium : contains

- 670 mg of yesst nitrogen base w/o AA

- 2g glucose (2%) or 10 ml glucose (20%)

- 200 mg AA mix

- 800ul of Uracile

- 800 ul of Leucine

- 800 ul of Histidine.

• M9/minimal + thiamine : contains

- 10 ml of M9 salts 10x

- 0.2 ml of MgSO4 (1M)

- 0.1 ml of CaCl2 (0.1 mM)

- 10 ml of glucose (20%) -> 2%

- 100 ul of thiamine (1mg/ml)

• M9/minimal + AA : contains

- 10 ml of M9 salts 10x

- 0.2 ml of MgSO4 (1M)

- 0.1 ml of CaCl2 (0.1 mM)

- 10 ml of glucose (20%) -> we actually now think it's better to put 4% so 20 ml

- 200 mg of AA mix

- 800 ul of uracile

- 800 ul of histidine

- 800 ul of leucine

• M9 salts 10x -> 500 ml

- 30 g Na2HPO4 * 7H2O

- 15 g KH2PO4

- 2.5 g NaCl

- 5 g NH4Cl

Culture

3x5ml of RFP (I13507)

3xRO2#11 (SC/M9+thia/M9+AA+thia)

3xRO2#5 (SC/M9+thia/M9+AA+thia)

3xRO2#10 (SC/M9+thia/M9+AA+thia)

People in the lab

Basile, Mélanie, Caroline