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| 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 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. |
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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.
|
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