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Team:Freiburg/Notebook/28 July
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==<span style="color:green;">green light receptor</span>== | ==<span style="color:green;">green light receptor</span>== | ||
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Incubation for 30 min 37°C.<br/> | Incubation for 30 min 37°C.<br/> | ||
Afterwards the digested product was loaded onto gel.<br/> | Afterwards the digested product was loaded onto gel.<br/> | ||
| - | Comments: | + | Comments: Not expected results. We will try a gradient PCR.<br/> |
<br/> | <br/> | ||
| - | [[File:29_07_Lov-tap.jpg]] | + | [[File:29_07_Lov-tap.jpg|250px|caption|border]] |
===Gradient PCR for Lov-tap S35=== | ===Gradient PCR for Lov-tap S35=== | ||
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*54-59°C | *54-59°C | ||
*64-72°C | *64-72°C | ||
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| + | After the PCR we will load the samples onto a gel to see the results of the gradient PCR.<br/>PCR did not work, only bands with 1000bp instead of 3000bp. We will order new Primers and then try again.<br/> | ||
| + | [[File:7_30_2011_1_45_01AM.jpg|caption|border]] | ||
===Miniprep of Not-Gate=== | ===Miniprep of Not-Gate=== | ||
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*3 μl DNA (M45a-M45d)<br/> | *3 μl DNA (M45a-M45d)<br/> | ||
| - | Incubation for 1 hour. Gel was loaded afterwards. | + | Incubation for 1 hour. Gel was loaded afterwards.<br/> |
| + | Comments: M45c and M45d look good. | ||
[[File:7_29_2011_9_52_26PM.jpg|caption|border]] | [[File:7_29_2011_9_52_26PM.jpg|caption|border]] | ||
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because there was some confusion with the labbeling and no stock, I am repeating transformation and DNA extraction. | because there was some confusion with the labbeling and no stock, I am repeating transformation and DNA extraction. | ||
| - | ==<span style="color: | + | ==<span style="color:grey;">Precipitator</span>== |
| - | === | + | ===Miniprep=== |
| - | '''Investigators: | + | '''Investigators: Rüdiger''' |
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| - | + | ||
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| - | + | ||
| - | + | Miniprep with Hiss-kit. Samples from yesterday were inoculated: | |
| + | *43-20 a-d | ||
| + | *43-19 a-d | ||
| + | *43-18 a,b | ||
| + | *39-19 | ||
| + | *39-20 | ||
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{{:Team:Freiburg/Templates/footer}} | {{:Team:Freiburg/Templates/footer}} | ||
Latest revision as of 01:02, 22 September 2011
Contact 
Contents |
green light receptor
Digest of Qickchange PCR product with DpnI
Investigators:Julia
in order to get rid of old plasmids a digest with DpnI was performed.
Transformation
Competent cells (50µl) were transformed with 10µl of the PCR- Product after iGEM Protocol.
CcaS is in the iGEM vector pSB1K3
CcaR in pSB1C3.
blue light receptor
Digestion of PCR product
Investigators:Sandra
Digest of PCR product S35 (Lov-tap), because PCR did not work out. We got a band of 1000bp, but we expected 3000bp.
Digest:
- 1 μl NEB4 buffer
- 1 μl BSA
- 1 μl NcoI
- 2 μl H2O
- 5 μl S35
Incubation for 30 min 37°C.
Afterwards the digested product was loaded onto gel.
Comments: Not expected results. We will try a gradient PCR.
Gradient PCR for Lov-tap S35
Investigators: Sophie
Comments: Because PCR for S35 did not work, we are doing a gradient PCR.
PCR as usual. Only temperature were adjusted, to determine the optimum of the annealing temperature.
- 54-59°C
- 64-72°C
After the PCR we will load the samples onto a gel to see the results of the gradient PCR.
PCR did not work, only bands with 1000bp instead of 3000bp. We will order new Primers and then try again.
Miniprep of Not-Gate
Investigators: Sophie
Picked colonies from plate and then performed a miniprep.
DNA-concentration measured with nanodrop:
| sample | DNA-concentration (ng/μl) |
| M45a | 248.9 |
| M45b | 256.8 |
| M45c | 291.3 |
| M45d | 295.8 |
Testdigest of Miniprep
Investigators: Sophie
Testdigest of M45a-M45d.
- 4 μl H20
- 1 μl NEB4
- 1 μl BSA
- 0.5 μl EcoRI
- 0.5 μl PStI
- 3 μl DNA (M45a-M45d)
Incubation for 1 hour. Gel was loaded afterwards.
Comments: M45c and M45d look good.
red light receptor
Miniprep of BBa_I15008 and BBa_I15009
Investigators:Julia
because there was some confusion with the labbeling and no stock, I am repeating transformation and DNA extraction.
Precipitator
Miniprep
Investigators: Rüdiger
Miniprep with Hiss-kit. Samples from yesterday were inoculated:
- 43-20 a-d
- 43-19 a-d
- 43-18 a,b
- 39-19
- 39-20
