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Team:DTU-Denmark/Notebook/15 July 2013
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{{:Team:DTU-Denmark/Templates/StartPage|15 July 2013}} | {{:Team:DTU-Denmark/Templates/StartPage|15 July 2013}} | ||
| - | + | Navigate to the [[Team:DTU-Denmark/Notebook/14_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/16_July_2013|Next]] Entry | |
| - | + | =Lab 208= | |
| - | + | <hr/> | |
| - | =208= | + | |
| - | + | ||
==Main purpose== | ==Main purpose== | ||
| + | <hr/> | ||
*Colony PCR of AMO, HAO, cytochromes and Nir transformants | *Colony PCR of AMO, HAO, cytochromes and Nir transformants | ||
==Who was in the lab== | ==Who was in the lab== | ||
| + | <hr/> | ||
Julia, Kristian, Gosia | Julia, Kristian, Gosia | ||
==Procedure== | ==Procedure== | ||
| + | <hr/> | ||
===Colony PCR=== | ===Colony PCR=== | ||
| - | [ | + | [[Team:DTU-Denmark/Methods/Colony_PCR|Colony PCR]] was performed according to standard method in order to check if transformed cells which grew on medium contain desired insert in pZA21 plasmid. |
| - | Constructs for transformations contained one gene in plasmid pZA21 and were created using USER reaction. | + | Constructs for transformations contained one gene in plasmid pZA21 and were created using USER reaction. Inserted genes are as follows: HAO, AMO, cycAX (cytochromes), Nir operon. |
Several colonies were chosen from each plate to be checked. | Several colonies were chosen from each plate to be checked. | ||
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* RFP 1 - RFP 20 - RFP in pZA21, primers 14a, 14b | * RFP 1 - RFP 20 - RFP in pZA21, primers 14a, 14b | ||
| - | PCR programms based on standard [ | + | PCR programms based on standard [[Team:DTU-Denmark/Methods/PCR|PCR program]] with differences in annealing temperatures and extension time as it was done on [[Team:DTU-Denmark/Notebook/12_July_2013|12-07-2013]]. |
===Gel electrophoresis=== | ===Gel electrophoresis=== | ||
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*10 mL Casamino Acid (from stock solution) | *10 mL Casamino Acid (from stock solution) | ||
| - | *2 mM | + | *2 mM MgSO<sub>4</sub> (1mL from 2M liquid stock) |
| - | *0.1 mM | + | *0.1 mM CaCl<sub>2</sub> (0.1mL from 1M liquid stock) |
*40g glucose (then the final solution will be 0.4 weight/vol) | *40g glucose (then the final solution will be 0.4 weight/vol) | ||
*200 mL M9 salts x5 | *200 mL M9 salts x5 | ||
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1L x5 stock of M9 salts: | 1L x5 stock of M9 salts: | ||
| - | * 64 g | + | * 64 g Na<sub>2</sub>HPO<sub>4</sub>*7H<sub>2</sub>O = 31.2 g Na2HPO4 |
| - | * 15 g | + | * 15 g KH<sub>2</sub>PO<sub>4</sub> |
* 25 g NaCl | * 25 g NaCl | ||
| - | *5 g | + | *5 g NH<sub>4</sub>Cl |
==Conclusion== | ==Conclusion== | ||
| + | <hr/> | ||
USER transformant from last Saturday didn't seem very good and we speculate that this could be because of membrane stress caused by all of the membrane proteins that we are suddenly incorporating. We will try to get around this by making a vector with an arabinose inducible promoter. Then there will be no damaging protein expression before we apply arabinose. | USER transformant from last Saturday didn't seem very good and we speculate that this could be because of membrane stress caused by all of the membrane proteins that we are suddenly incorporating. We will try to get around this by making a vector with an arabinose inducible promoter. Then there will be no damaging protein expression before we apply arabinose. | ||
| - | |||
Navigate to the [[Team:DTU-Denmark/Notebook/14_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/16_July_2013|Next]] Entry | Navigate to the [[Team:DTU-Denmark/Notebook/14_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/16_July_2013|Next]] Entry | ||
{{:Team:DTU-Denmark/Templates/EndPage}} | {{:Team:DTU-Denmark/Templates/EndPage}} | ||
Latest revision as of 11:39, 4 October 2013
15 July 2013
Contents |
Lab 208
Main purpose
- Colony PCR of AMO, HAO, cytochromes and Nir transformants
Who was in the lab
Julia, Kristian, Gosia
Procedure
Colony PCR
Colony PCR was performed according to standard method in order to check if transformed cells which grew on medium contain desired insert in pZA21 plasmid. Constructs for transformations contained one gene in plasmid pZA21 and were created using USER reaction. Inserted genes are as follows: HAO, AMO, cycAX (cytochromes), Nir operon.
Several colonies were chosen from each plate to be checked. Names and numbers of colonies as well as plates names and primers are:
- 1,2 - HAO in pZA21 USER, primers 18a, 18b
- 3-10 - AMO in pZA21 USER, primers 17a, 17b
- 11-24 - cycAX in pZA21 USER, primers 16a, 16b
- 25-28 - Nir in pZA21 USER, primers 15a, 15b
- RFP 1 - RFP 20 - RFP in pZA21, primers 14a, 14b
PCR programms based on standard PCR program with differences in annealing temperatures and extension time as it was done on 12-07-2013.
Gel electrophoresis
Samples after colony PCR were checked on 1% agarose gel today and most will be checked tomorrow.
M9
Made 3L of M9 minimal medium the the following recipe for 1L of medium:
- 10 mL Casamino Acid (from stock solution)
- 2 mM MgSO4 (1mL from 2M liquid stock)
- 0.1 mM CaCl2 (0.1mL from 1M liquid stock)
- 40g glucose (then the final solution will be 0.4 weight/vol)
- 200 mL M9 salts x5
- Fill to 1 L with sterilized water
1L x5 stock of M9 salts:
- 64 g Na2HPO4*7H2O = 31.2 g Na2HPO4
- 15 g KH2PO4
- 25 g NaCl
- 5 g NH4Cl
Conclusion
USER transformant from last Saturday didn't seem very good and we speculate that this could be because of membrane stress caused by all of the membrane proteins that we are suddenly incorporating. We will try to get around this by making a vector with an arabinose inducible promoter. Then there will be no damaging protein expression before we apply arabinose.
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