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From 2010.igem.org

8/23/2010

Cold shock results:
One red colony on IPTG plate at 4C (first observed at 9:20 am on 8/20/2010) and one red colony observed on IPTG at 15C (8/23/2010).

8/23/2010

Purpose: try to maximize expression of mRFP.
Making a “starter” culture:
1. From 4C NON-IPTG plate, take a white colony and place in approx. 3mL of LB/CARB media.
2. Incubate with shaking overnight at 37C.

8/24/2010
Purpose: from starter culture monitor OD to obtain OD600 of 0.4-0.5.

1. From starter culture take 50mL liquid culture and add 50uL CARB in a large Erlenmeyer flask.
2. To this, add 1mL from starter culture and begin monitoring OD (for each aliquot take 1mL for measuring OD); maintain flask at 37C incubation with shaking. Keep in mind that e-coli doubling time is 20 minutes.
3. Put on incubator/shaker at 11:20 am, with initial total volume of 51mL, and OD600 = 0.0.

aliquot	volume remaining in flask	time	OD600
1	50mL	11:45am	0.08
2	49mL	12:45pm	0.32
3	48mL	1:00pm	0.56

4. Put incubator at 15C. During the cooling period, the flask was kept at 6C to ensure that the colony quit growing. Final OD was 0.57 before putting in the incubator (without shaking) for 30 minutes. (Time in: 1:30 pm). Total volume of the flask at this point was 47mL.
5. At 2:00 pm, remove flask from incubator and IPTG (100mM) to a final concentration between 0.1 and 1mM (choose 0.25 mM).  For a final concentration of 0.25mM IPTG in a 47mL solution, add 117.5uL of IPTG (100mM stock).
6.  Continue culture with shaking at 15C for 24 hours.

8/25/2010

Results from 8/24/2010: expression unsuccessful.
Start with fresh colonies from the plates from 8/19/2010.  Since we know the mRFP made it successfully into the pCold vector on the two colonies that turned red, try to make starter cultures from those.

Making a “starter” culture:
1. From 4C and 15C IPTG plate, take a red colony and place in approx. 3mL of LB/CARB media. (3mL LB + 3uL CARB).
2. Incubate the two tubes with shaking overnight at 37C.

Plan for 8/26/2010: Make cultures as we did on 8/24/2010 again, monitoring the concentration of cells.  Then run a miniprep to ensure that we have the mRFP inserted into the pCold vector.

8/26/2010

Successful growth of starter colonies. Repeat process from 8/24/2010 until cell concentration is OD600 = 0.4 to 0.5.

1. Prepare 2 flasks (1 for each starter colony) acccording to directions from 8/24/2010.
2. Repeat procedure from 8/24/2010. (final concentration of cells in each flask was approx. OD600 = 0.4). Amount of (100mM) IPTG added: 125uL to each flask.
3. Incubate with shaking overnight at 15C.

8/27/2010

WE GOT RED FLASKS!!!!!!!!!!!!

Now, we’re going to do a Miniprep and run an experiment analogous to what we did on 8/19/2010.

Miniprep of 2 cultures: one inoculated from a fluorescent colony on the 4º C plate; one inoculated from a fluorescent colony on the 15º C. 

1. Protocol from 8/12/2010
2. Concentrations:

pCOLD+mRFP (4º C)	41.2 ng/uL
pCOLD+mRFP (15º C)	67.1 ng/uL

Transformation of BL21 by pCOLD+mRFP (from 4C  and 15ºC cultures)

1. Plasmid, cells should be on ice initially
2. + 1uL plasmid into 20 uL of competent cells (BL21)
3. Sit on ice, 10 mins
4. place in 42º water bath, 30 seconds
5. place in ice, 2 minutes
6. +250 uL LB broth
7. Plate: spread 100 uL onto plate, use spatula to spread around until resistance is felt.
1. I plated an IPTG plate and a non-ITPG plate
2. The IPTG plate had 25 uL of 0.1 M IPTG
8. Plates were left (upside down) on bench at room temperature (since the starter culture is not needed until Sunday, and these cells were transformed today, Friday)

For Next Time:
Sunday: Start 5 mL culture with one fluorescent colony of pCOLD+mRFP
Monday: Protein Expression under various conditions (i.e. cold, iptg, cold+iptg) in 5x50mL flasks.