EPF-Lausanne/3 October 2009

Wet Lab

Results of yesterday's culture

DH5a :

-RO1.1 + BB1 /#1 in LB and in M9 grew.

-RO2.4 + BB1 /#3 in LB grew (and were redish) and in M9 grew as well.

-RO1.1 + BB1 /#1 (JW strain) LB -> grew, and were redish

-RO1.1 + BB1 /#2 (JRG 1046) LB -> grew, redish too.

They all grew (they were taken from the glycerol stock) but some didn't have the colour they should have. They are all medium colour but the :

-RO2.4 + BB1 (LB) should be redish

-Both RO1.1 + BB1 in K.O. strains should also be redish.

So we are going to do a colony PCR to see if the plasmids are there.

Colony PCR

We used the Taq platinium protocol. No volume added for template DNA : just pelleted cells.

1. RO1.1 + BB1/#1 (DH5a) "in LB"

2. RO1.1 + BB1/#1 (DH5a) "in M9"

3. RO2.4 + BB1/#3 (DH5a) "in LB"

4. RO2.4 + BB1/#3 (DH5a) "in M9"

5. RO1.1 + BB1/#1 (JW) "in LB"

6. RO1.1 + BB1/#2 (JRG 1046) "in LB"

7. Positive control : from 1 ul plasmid RO1,3

8. Negative control : no DNA.

Template : from pelleted cells from 500 ul of the overnight culture.

Characterization

Setting the conditions for culture

For the qPCR.

For all 6 strains /double transformants / medium. Different conditions : +/- IPTG, +/- light, +/- TRP.

DH5a : RO1.1 + BB1/#1 (LB & M9), RO2.4 + BB/#3 (LB & M9)

JW : RO1.1 + BB1 /#1 (LB)

JRG 465 : RO1.1 + BB1 /#2 (LB).

The cells / conditions without light were put in culture tubes and then in the aluminium box.

The cells / conditions with light were put in 50 ml flasks and then covered with desinfected parafin foil (instead of aluminium foil in order to let the light shine right on the cells).

We put them in Sebastian's incubator at 37°C and 200 rpm shaking for about 2hours.

Loading the plate

In order not to activate the cells that have been grown without light, we used the following method :

- we went to a dark room

- we used the red LEDs to see what we were doing

- we loaded the plate

20 ul of corresponding fresh medium + 40 ul of cells/condition loaded in each well.

-> qPCR machine at Sebastian's lab : 40 cycles of 3 minutes at 37°C.

Transformation

These 3 strains will be transformed :

- DH5a

- JRG1046

- JRG4 465

Note : JW has been abandoned for now as it seems not to retain the BB (Kana) plasmid.

DNA volume for the double transformation

RO2.4 6.02 ul

BB1 3.98 ul (if put with RO2.4) and 5.66 ul (if put with RO1.1)

RO1.1 4.34 ul

Gel

We did a gel after the colony PCR of this morning.

The gel was not convincing, a new gel will be done tomorroww (this one seems "curved" so difficult to analyze).

Replating

We re-plated (for dilution) :

RO2.4 + BB1 JRG 465

RO2.4 + BB1 JW 4356-2

All of this will be replated once more (still no single colonies).

Then we did a colony PCR of replated RO2.4 + BB1 in both JW 4356-2 and JRG 465 strains. Clones that were chosen were replated.

People in the lab

Nath, Basile