Team:Freiburg/Notebook/27 July

From 2011.igem.org

(Difference between revisions)
(→Gel)
 
(23 intermediate revisions not shown)
Line 1: Line 1:
 +
{{:Team:Freiburg/Templates/header}}
 +
<html>
 +
<div id="notebook-page-header">
 +
<div id="notebook-back" width="100px" >
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook/26_July">Previous entry</a>
 +
</div>
 +
<div id="notebook-title">
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 27 July </a>
 +
</div>
 +
<div id="notebook-next">
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook/28_July">Next entry</a>
 +
</div>
 +
</div>
 +
</html>
 +
==Commons==
==Commons==
===Gel===
===Gel===
Line 4: Line 19:
'''Investigators: Sandra, Sophie'''
'''Investigators: Sandra, Sophie'''
-
We loaded the PCR products of the vectors on a gel and measured the DNA concentration.
+
We loaded the PCR products of the vectors on a gel and measured the DNA concentration. DNA concentration were high and we diluted the vectors to get an endconcentration of 25ng/microl.
 +
 
 +
'''DNA-concentration measured with nanodrop:'''<br/>
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|sample
 +
|DNA concentration (ng/μl)
 +
|-
 +
|pSB1C3
 +
|132.1
 +
|-
 +
|pSB1K3
 +
|101.5
 +
|-
 +
|pSB1A3
 +
|83.9
 +
|-
 +
|pSB1T3
 +
|116.2
 +
|}
 +
 
 +
 
 +
[[File:.Gelbild_Vektoren_27_07.jpg|350px|caption]]
==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Results of Sequencing===
 +
 
 +
'''Investigators:Julia'''
 +
 
 +
 
 +
CcaS 4a2 seemed to miss about 20bp, CcaS 4b2 is correct.
 +
 
 +
===Quickchange PCR of CcaS and CcaR===
 +
 
 +
'''Investigators:Julia'''
 +
 
 +
in order to eliminate iGEM restriction sites of EcoRI within CcaS and CcaR, we designed mutagenesis primer and performed a quickchange PCR.
 +
 
 +
===Protocol===
 +
 
 +
total reaction volume is 50µl:
 +
 
 +
32,5 µl H2O<br/>
 +
10 µl Phusion Buffer 5x<br/>
 +
2,5 µl Primer up<br/>
 +
2,5 µl Primer dw<br/>
 +
1 µl DNA template, concentration has to be 5ng/µl<br/>
 +
0,5 µl Phusion<br/>
-
'''Investigators:NAME'''
+
Primer for CcaR:<br/>
 +
ccaR_mut1_up: CCtcactaATGAGgATcCTTTTAGTGGAGG<br/>
 +
ccaR_mut1_dw:CCTCCACTAAAAGgATcCTCATtagtgaGG<br/>
 +
<br/>
 +
Primer for CcaS:<br/>
 +
ccaS_mut1_up:CCGGGCCGAGgATcCTCTATGTCAATG<br/>
 +
ccaS_mut1_dw:CATTGACATAGAGgATcCTCGGCCCGG<br/>
 +
<br/>
 +
Primer for CcaS:<br/>
 +
ccaS_mut2_up:GGACCAAAAACgAGTCGCACTGAATTAG<br/>
 +
ccaS_mut2_dw:CTAATTCAGTGCGACTcGTTTTTGGTCC<br/>
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
Line 20: Line 88:
We repeated the PCR again, with higher temperatures this time.
We repeated the PCR again, with higher temperatures this time.
After PCR we digested with DpnI 37°C for 1 hour and then purified the DNA with PCR purifictaion kit. Afterwards we measured the DNA concentration and loaded 2 microl to a gel.
After PCR we digested with DpnI 37°C for 1 hour and then purified the DNA with PCR purifictaion kit. Afterwards we measured the DNA concentration and loaded 2 microl to a gel.
 +
'''PCR'''
'''PCR'''
Line 31: Line 100:
|-
|-
-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment: NOT-Gate and LovTAP with Gibson-overhangs (now we use a little bit higher temperature for primer annealing) (Date): 25.07.11
+
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment: NOT-Gate and LovTAP with Gibson-overhangs (now we use a little bit higher temperature for primer annealing) -(Date): 25.07.11
-
(Name): Blue Light
+
(Name): Sandra, Sophie
|-
|-
-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
+
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Blue Light
|}
|}
Line 88: Line 157:
What program do you use?
What program do you use?
-
57c auf 70c (in reality: 58c and then 68c)
+
"57°C auf 70°C" (first annealing temperature: 58°C and then after 10 cycles 68°C)
Line 96: Line 165:
How did you label the PCR-Product, where is it stored and what do you do next?
How did you label the PCR-Product, where is it stored and what do you do next?
-
==<span style="color:red;">red light receptor</span>==
 
-
===NAME OF YOUR EXPERIMENT===
+
'''DNA-concentration measured with nanodrop:'''
-
'''Investigators:NAME'''
+
{| cellpadding="10" cellspacing="0" border="1"
 +
|sample
 +
|DNA-concentration (ng/μl)
 +
|-
 +
|S35
 +
|75.1
 +
|-
 +
|S45
 +
|45.1
 +
|}
 +
==<span style="color:grey;">Precipitator</span>==
-
==<span style="color:orange;">Lysis cassette</span>==
+
Ruediger  27.07
 +
picked colonies from yesterdays Trafo into medium with 2μl/ml Tet
-
===NAME OF YOUR EXPERIMENT===
+
S39-P18
-
 
+
S39-P19
-
'''Investigators:NAME'''
+
S39-P20
-
 
+
-
 
+
-
 
+
-
==<span style="color:grey;">Precipitator</span>==
+
-
===NAME OF YOUR EXPERIMENT===
+
S43-P18
 +
S43-P19
 +
S43-P20
-
'''Investigators: NAME'''
+
overnight in 37°C

Latest revision as of 01:01, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

Commons

Gel

Investigators: Sandra, Sophie

We loaded the PCR products of the vectors on a gel and measured the DNA concentration. DNA concentration were high and we diluted the vectors to get an endconcentration of 25ng/microl.

DNA-concentration measured with nanodrop:

sample DNA concentration (ng/μl)
pSB1C3 132.1
pSB1K3 101.5
pSB1A3 83.9
pSB1T3 116.2


caption

green light receptor

Results of Sequencing

Investigators:Julia


CcaS 4a2 seemed to miss about 20bp, CcaS 4b2 is correct.

Quickchange PCR of CcaS and CcaR

Investigators:Julia

in order to eliminate iGEM restriction sites of EcoRI within CcaS and CcaR, we designed mutagenesis primer and performed a quickchange PCR.

Protocol

total reaction volume is 50µl:

32,5 µl H2O
10 µl Phusion Buffer 5x
2,5 µl Primer up
2,5 µl Primer dw
1 µl DNA template, concentration has to be 5ng/µl
0,5 µl Phusion

Primer for CcaR:
ccaR_mut1_up: CCtcactaATGAGgATcCTTTTAGTGGAGG
ccaR_mut1_dw:CCTCCACTAAAAGgATcCTCATtagtgaGG

Primer for CcaS:
ccaS_mut1_up:CCGGGCCGAGgATcCTCTATGTCAATG
ccaS_mut1_dw:CATTGACATAGAGgATcCTCGGCCCGG

Primer for CcaS:
ccaS_mut2_up:GGACCAAAAACgAGTCGCACTGAATTAG
ccaS_mut2_dw:CTAATTCAGTGCGACTcGTTTTTGGTCC

blue light receptor

PCR of Lov-tap and Not-gate

Investigators: Sandra, Sophie

We repeated the PCR again, with higher temperatures this time. After PCR we digested with DpnI 37°C for 1 hour and then purified the DNA with PCR purifictaion kit. Afterwards we measured the DNA concentration and loaded 2 microl to a gel.

PCR


Name:Sandra, Sophie


Date: 27.07.11
Continue from Experiment: NOT-Gate and LovTAP with Gibson-overhangs (now we use a little bit higher temperature for primer annealing) -(Date): 25.07.11

(Name): Sandra, Sophie

Project Name: Blue Light

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 5x Phusion Buffer of Primer
2.5µl Primer fw NOT_G_up

LOV_G_up

2.5µl Primer dw NOT_G_dw

LOV_G_dw

1µl dNTPs of Template DNA
1µl DNA-Template S35 (BBa_K322999)

S45 (Bba_Q04400)

0.5 µl Phusion (add in the end)

What program do you use?

"57°C auf 70°C" (first annealing temperature: 58°C and then after 10 cycles 68°C)


To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.


How did you label the PCR-Product, where is it stored and what do you do next?


DNA-concentration measured with nanodrop:

sample DNA-concentration (ng/μl)
S35 75.1
S45 45.1


Precipitator

Ruediger 27.07 picked colonies from yesterdays Trafo into medium with 2μl/ml Tet

S39-P18 S39-P19 S39-P20

S43-P18 S43-P19 S43-P20

overnight in 37°C

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/27_July"