Team:UTP-Panama/After Regional Week 1

From 2011.igem.org

(Difference between revisions)
(October 19th)
(Octuber 20)
 
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|-
|-
| -
| -
-
|genetic material from minipreparations
+
|genetic material from MiniPrep
|-
|-
|?
|?
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|-
|-
|2
|2
-
|tubes with cloramphenicol (liquid or powder)
+
|tubes with chloramphenicol (liquid or powder)
|-
|-
|2
|2
Line 40: Line 40:
|-
|-
|1 jar
|1 jar
-
|MgCl2 (for doing comp. cells)
+
|MgCl2 (for making Competent Cells)
|-
|-
|1 jar
|1 jar
-
|CaCl2 (for doing comp. cells)
+
|CaCl2 (for making Competent Cells)
|-
|-
|1 jar
|1 jar
Line 49: Line 49:
|-
|-
|3 jars
|3 jars
-
|minipreparation Buffers (P1,P2,P3)
+
|MiniPrep Buffers (P1,P2,P3)
|-
|-
|3 boxes
|3 boxes
Line 69: Line 69:
<br>- antibiotics
<br>- antibiotics
<br>- LB media
<br>- LB media
-
<br>- Eppendorf 2 mL for auto-nailing
+
<br>- Eppendorf 2 mL for autoclaving
<br><br>
<br><br>
Line 89: Line 89:
We had to:  
We had to:  
<br>- prepare new antibiotics
<br>- prepare new antibiotics
-
<br>- auto-nail the eppendorf of 2 mL, they were ready.
+
<br>- autoclave the eppendorfs of 2 mL, they were ready to autoclave.
<br>- prepare the Minimal Media for the experiments.
<br>- prepare the Minimal Media for the experiments.
<br><br>
<br><br>
For the new experiments:
For the new experiments:
-
<br>- we used 5 mL of sustrates (liquid LB and MME)
+
<br>- we will use 5 mL of sustrates (liquid LB and MME)
<br>- We did controls with and wothout antibiotics
<br>- We did controls with and wothout antibiotics
<br>- We used the following colonies:
<br>- We used the following colonies:
Line 116: Line 116:
<br>  - measurement of heat production (thermometer)
<br>  - measurement of heat production (thermometer)
-
<div align="center">Experiments:
+
<div align="center">'''Experiments:'''</div>
<br>
<br>
<br>#1: 5 colonies in LB and MM in blank, for references without antibiotics and without nitrate, @37ºC and @18ºC.
<br>#1: 5 colonies in LB and MM in blank, for references without antibiotics and without nitrate, @37ºC and @18ºC.
Line 124: Line 124:
<br>#5: 5 colonies in LB and MM with antibiotics and nitrate @37ºC, first passing through thermal shock and @37ºC without thermal shock, direct growing.
<br>#5: 5 colonies in LB and MM with antibiotics and nitrate @37ºC, first passing through thermal shock and @37ºC without thermal shock, direct growing.
<br><br>
<br><br>
-
For Thursday, October 20th,  2011:
+
Things we have to do for tomorrow, Thursday, October 20th,  2011:
<br>- Transforms competent cells with:
<br>- Transforms competent cells with:
<br>  - Bristol  
<br>  - Bristol  
<br>  - Renbo, ligated with ampicillin
<br>  - Renbo, ligated with ampicillin
<br><br>
<br><br>
-
For Friday, October 21st, 2011:
+
Things we have to do for Friday, October 21st, 2011:
<br>  - take the plates out from the incubator
<br>  - take the plates out from the incubator
<br>  - grow a Renbo colony, in liquid LB @37ºC with shaker.
<br>  - grow a Renbo colony, in liquid LB @37ºC with shaker.
<br><br>
<br><br>
-
For Saturday, October 22nd, 2011:
+
Things we have to do for Saturday, October 22nd, 2011:
<br>  - miniprep. of Renbo
<br>  - miniprep. of Renbo
<br>  - electrophoresis of Renbo.
<br>  - electrophoresis of Renbo.
 +
 +
===Octuber 20===
 +
<br>
 +
Bristol (choromphenicol)<br>
 +
Rembo (Ampycillin)<br>
 +
We used 4 plates for each ,renbo and Bristol:<br>
 +
First plate( LB ,antibiotic ,renbo) <br>
 +
Second plate(LB, antibiotic, competent cells without plates)<br>
 +
Third plate( LB,  competent cells)<br>
 +
Forth plate(Thermal  shock at 45°C for 1 minute)<br>
 +
4 parts of 500µl of competent cells<br>
 +
Using the protocol of page 40 but instead using 20ml of solid LB.<br>
 +
RENBO=  30 µl of ampycillin<br>
 +
BRISTOL=14.6 µL of cloromphenicol<br>
 +
 +
Observations<br>
 +
1) We make calculations for 15 ml of lb .<br>
 +
2) For  the negative control ( LB + Competent cells ) we just use 1 plate,  so in the incubator we had 7 plates.
 +
3 for Renbo <br>
 +
#1(LB+ ampicillin+ competent cells)(control)<br>
 +
#2(LB+ampicillin+renbo)<br>
 +
#3(LB+ampicillin+renbo)<br>
 +
<br>
 +
3 for Bristol<br>
 +
#1(LB+ clorophenicol+ competent cells)(control)<br>
 +
#2(LB+clorophenicol+bristol)<br>
 +
#3(LB+clorophenicol+bristol)<br>
 +
 +
1 plate generally negative<br>
 +
(LB + Competent cells) <br>

Latest revision as of 03:56, 29 October 2011

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October 17 to 23

October 18th

INVENTORY:

Quantity Description
24 petri plates (60 x 15 mm)
a lot! eppendorf tubes of 0,5 mL
4 tubes with linearized plasmids (kit)
8 tubes with competent cells
- genetic material from MiniPrep
? ligation kit
2 tubes with SOC media (15 mL)
2 tubes with chloramphenicol (liquid or powder)
2 petri dishes with GaTech colonies
1 petri plates with RFP colonies
1 jar MgCl2 (for making Competent Cells)
1 jar CaCl2 (for making Competent Cells)
1 jar with ultrapure water
3 jars MiniPrep Buffers (P1,P2,P3)
3 boxes small pipette tips (10 uL)
4 boxes medium pipette tips (200 uL)
3 boxes large pipette tips (1000 uL)
35 falcon tubes of 15 mL

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)

Experiments:



#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

(LB + Competent cells)