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From 2010.igem.org

8/16/2010

Inoculation of cell cultures 5, 8 and 9

1. In a Eppendorf tube add 3սL carbinicillin (CARB) to 3 mL LB media.
2. Inoculate at 37C overnight.

 

8/17/2010

Cell culture tubes 5, 8, and 9 from 8/16/2010 were removed from inoculation and used for two purposes:

Freezing a portion of inoculated cells for future use
300 սL of glycerol were added to 200 սL of each culture and tapped gently until the culture had mixed with the glycerol. These were then marked and stored in the freezer in the upstairs lab.

Addition of IPTG to induce expression of pCOLD
To test the effects of IPTG on mRFP (and thus pCOLD) expression, we prepared 4 concentrations of IPTG for each of the 3 tubes of bacteria (5, 8, and 9), resulting in 12 tubes.

Each contained:
930սL LB media
50սL culture
10սL CARB
10սL IPTG

where the 10սL of IPTG were calculated serial dilutions insured to give an overall IPTG concentration of 0.1 M (10^-3), 1 mM (10^-5), 10 սM (10^-7), and 100 nM (10^-9) for each tube number 5, 8, and 9. The resulting 12 samples were put in the incubator at 37C overnight.

If everything goes according to plan, it should be possible to see some RFP expression tomorrow.

8/18/2010

Expression of mRFP should have been detectable by red coloring in the samples after incubation.  Nothing was seen, and as such the samples were discarded. WE PUT THEM IN THE WRONG CELLS! Novablue cells are inapropriate for this type of transformation... redo the transformation with BL21 cells.

8/19/2010
Transformation into BL21 cells

Adding IPTG to plates:
1.  Spread 50սL IPTG on 2 LB/carb plates (50սL each plate).
2. Wait 1 hour to allow IPTG to soak into media.

Preparing cells:
3. Chill 2 empty 1.5սL Eppendorf tubes, 1:4 ligation reaction product, and 20սL BL21 cells on ice.
4. Aliquot 10սL of BL21 cells into Eppendorf tubes (10սL each tube).
5. In each tube add 1սL 1:4 ligation reaction mixture.
6. Allow to sit on ice for 10-30 min.
7.  Place in 42C bath for 30-45 sec.
8. Immediately place back on ice for 2 min.
9. Add 250սL room temp. LB media to reaction tube.
10.  Places tubes in 37C incubator/shaker for 1 hr.
11. Spread 125սL of incubated cells onto labelled LB/CARB plates according to below:

Plate	IPTG	CELLS	TEMP
1	50սL	125սL	4C
2	50սL	125սL	15C
3	NONE	125սL	4C
4	NONE	125սL	15C

12. Incubate overnight (16 hrs) at 37C.

8/20/2010

Cold shock:
1. Place places in appropriate temperature incubators (4C or 15C), and wait to see expression of RFP.

Results: One red colony observed on IPTG plate at 4C at 9:20 am.