• July
• August
• September

July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
4. Waiting 30 min in ice
5. Heat shock 30 sec 42°C
6. Then 5min in ice
7. Add 200uL SOC medium
8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control

I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.

July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method

The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)

The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.

In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)

Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.

When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.

Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.

Centrifuge 10min at 4°C.

Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.

Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.

July 30th

Goal 1

PCR my G block and backbone

Procedure

PCR1 = Backbone + 068 and 069 primers, before purify

PCR2 = FINAL + 068 and 069 primers, before purify

Then PCR purification

Nanodrop : PCR 1 = 61ng/uL ; PCR2 = 25,4 ng/uL

Goal 2

Digestion/Ligation/Transformation

Procedure

First, digestion :

PCR1 ADN : 40uL = 2,4 ug 4uL EcoRI, PstI, FastAp 12 uL FD buffer 56 uL water TOT =120uL

PCR2 ADN : 40uL = 1ug 6 uL EcoRI, PstI 12 uL FD buffer 64 uL water TOT= 128uL

10 min at 37°C

Then PCR purification

Nanodrop : nanodrop : PCR 1 = 20,7 ng/uL ;PCR2 = 25,7

For the ligation :

Vector PCR1 100 ng = 5 uL; Insert PCR 2 300 ng = 12uL T4 ligase buffer 2uL ; T4 ligase 1 uL ; Total 20 uL

20-40 min on the bench

Nanodrop : L= 270 ng/uL

Then I did a heat shock transformation (following the iGEM protocol : http://parts.igem.org/Help:Protocols/Transformation )

C + = 10 ng/uL of RFP in pS1C3, 2 uL transformed (10pg/ul RFP Control (pSB1C3 w/ BBa_J04450)

C - = nothing but efficients cells

T = Plasmid construct 2uL transformed

Each with 50 uL of chemically competents cells done yesterday.

Then I did 5 different plates :

5 plates with 25ug/mL chloramphenicol in LB

200 uL control + , 20 uL control +

200 uL T , 20 uL T

200 uL C -

Incubation overnight at 37°C