Team:UTP-Panama/After Regional Week 1

October 17 to 23

October 18th

INVENTORY:

Missing:
- large gloves
- falcon tubes 50 mL
- antibiotics
- LB media
- Eppendorf 2 mL for autoclaving

October 19th

Ricardo reviewed the inventory and provided us:
- 1 box of large gloves
- 1 bag with falcons 50 mL
- 1 bag with eppendorf of 2 mL

Also:
- solid LB works
- not necessary to do liquid LB (there is)
- we have to transform competent cells with:
- Bristol (the plate isn't so good)
- RENBO (the product was ligated in the ampicillin plasmid) --> to do minipreparation and electrophoresis
- Colonies only without plasmids.

Plates with RFP and GaTech are fine!

We had to:
- prepare new antibiotics
- autoclave the eppendorfs of 2 mL, they were ready to autoclave.
- prepare the Minimal Media for the experiments.

For the new experiments:
- we will use 5 mL of sustrates (liquid LB and MME)
- We did controls with and wothout antibiotics
- We used the following colonies:
- JM109 (just the strain without plasmids)
- with RFP
- Bristol
- GaTech
- Renbo
- We did controls using the two methods:
- Direct, studying the temperature
- passing through the thermal shock, first.
- Thermic shock was:
- @10ºC per 1 hour
- Temperatures we used are:
- 37ºC
- between 15ºC and 20ºC (18ºC)
- We needed 5 days of experimentation.

for each temperature and BioBrick we wanted to get (en each experiment):
- growing curve
- measurement of GFP expression
- measurement of RFP expression, as control
- measurement of heat production (thermometer)

#1: 5 colonies in LB and MM in blank, for references without antibiotics and without nitrate, @37ºC and @18ºC.
#2: 5 colonies in LB and MM with nitrate @37ºC and @18ºC.
#3: 5 colonies in LB and MM with antibiotics @37ºC and @18ºC.
#4: 5 colonies in LB and MM with antibiotics and nitrate @18ºC, first passing through thermal shock and @18ºC without thermal shock, direct growing.
#5: 5 colonies in LB and MM with antibiotics and nitrate @37ºC, first passing through thermal shock and @37ºC without thermal shock, direct growing.

Things we have to do for tomorrow, Thursday, October 20th, 2011:
- Transforms competent cells with:
- Bristol
- Renbo, ligated with ampicillin

Things we have to do for Friday, October 21st, 2011:
- take the plates out from the incubator
- grow a Renbo colony, in liquid LB @37ºC with shaker.

Things we have to do for Saturday, October 22nd, 2011:
- miniprep. of Renbo
- electrophoresis of Renbo.

Octuber 20

Bristol (choromphenicol)
Rembo (Ampycillin)
We used 4 plates for each ,renbo and Bristol:
First plate( LB ,antibiotic ,renbo)
Second plate(LB, antibiotic, competent cells without plates)
Third plate( LB, competent cells)
Forth plate(Thermal shock at 45°C for 1 minute)
4 parts of 500µl of competent cells
Using the protocol of page 40 but instead using 20ml of solid LB.
RENBO= 30 µl of ampycillin
BRISTOL=14.6 µL of cloromphenicol

Observations
1) We make calculations for 15 ml of lb .
2) For the negative control ( LB + Competent cells ) we just use 1 plate, so in the incubator we had 7 plates. 3 for Renbo

1. 1(LB+ ampicillin+ competent cells)(control)
   
2. 2(LB+ampicillin+renbo)
   
3. 3(LB+ampicillin+renbo)
   

3 for Bristol

1. 1(LB+ clorophenicol+ competent cells)(control)
   
2. 2(LB+clorophenicol+bristol)
   
3. 3(LB+clorophenicol+bristol)
   

1 plate generally negative