Team:Freiburg/Notebook/14 July

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<a href="https://2011.igem.org/Team:Freiburg/Notebook/13_July">Previous entry</a>
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 14 July </a>
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==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
Line 10: Line 22:
3A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)
3A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)
-
S21a:
 
-
*Enzyme 1: XbaI
 
-
*Enzyme 2: PstI
 
-
S22, S23
+
'''Digestion'''
-
*Enzyme 1: EcoRI
+
-
*Enzyme 2: SpeI
+
-
Vector:
+
 
-
*Enzyme 1: EcoRI
+
{| style="border-spacing:0;"
-
*Enzyme 2: PstI
+
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Name:Jakob
-
*Enzyme 3: DpnI
+
| style="border:0.0139in solid #808080;padding:0.0194in;"| Date: 14.07.2011
-
<br/>
+
 
 +
|-
 +
| colspan="2"  style="border:0.0139in solid #808080;padding:0.0194in;"| Project Name: 3A-assembly of pcyA+ terminator, ho1+terminator
 +
 
 +
|}
 +
&nbsp;
 +
 
 +
 
 +
Procedure
 +
 
 +
&nbsp;
 +
 
 +
1. add H2O (16μl-DNA )&nbsp;&nbsp;&nbsp;
 +
 
 +
2. 2μl NEB4 buffer
 +
 
 +
3. 2 μl 10x BSA &nbsp;&nbsp; &nbsp;(used 1:10 diluted )
 +
 
 +
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
 +
 
 +
5. 1 μl restriction enzymes
 +
 
 +
6. heat for 1-2 hours 37°C (6 hours if time)
 +
 
 +
7. heat for 20 minutes 80°C (inactivation of enzymes)
 +
 
 +
8. keep at 4°C if you cannot continue
 +
 
 +
&nbsp;
 +
 
 +
Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Sample Name
 +
| style="border:0.0139in solid #808080;padding:0.0194in;"| DNA concentration (μg/μl)
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Vector&nbsp; pSB1T3
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 20
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| terminator
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 38.8
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| ho1
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 176
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| pcyA
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 173
 +
 
 +
|}
 +
&nbsp;
 +
 
 +
Restriction enzymes you need to cut the vector, insert1 and insert 2:
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| '''Components'''
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| <center>'''Vector (μl)'''</center>
 +
| colspan="2"  style="border:0.0139in solid #808080;padding:0.0194in;"| <center>'''Insert1 and 2 (μl)'''</center>
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| DNA (μl)
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 2
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Ho1 2.5
 +
 
 +
pcyA 2.5
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| Terminator 5.5
 +
 
 +
terminator 5.5
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| BSA (10x) (2μl)
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| NEB4 Buffer (2μl)
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Enzyme 1 (1μl)
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;EcoRI
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;EcoRI
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;XbaI
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Enzyme 2 (1μl)
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;PstI
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;SpeI
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;PstI
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| H2O (14 μl- DNA)
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| In total 20 μl
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
 +
 
 +
|}
 +
&nbsp;
===3A-assembly: Ligation and transformation===
===3A-assembly: Ligation and transformation===
Line 32: Line 150:
L23: Ligation of S23+S21a in pSB1T3
L23: Ligation of S23+S21a in pSB1T3
L22: Ligation of S22+S21a in pSB1T3
L22: Ligation of S22+S21a in pSB1T3
 +
'''Ligation'''
-
Plasmids were transformed in cells and plated out.
 
 +
{| style="border-spacing:0;"
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Name:Julia
 +
| style="border:0.0139in solid #808080;padding:0.0194in;"| Date:14.07
 +
 +
|-
 +
| colspan="2"  style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| Continue from&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Date14.07&nbsp;Name
 +
 +
Experiment&nbsp; &nbsp;&nbsp;&nbsp;3A-assembly
 +
 +
|-
 +
| colspan="2"  style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| Project Name:3A-assembly of pcyA+ terminator, ho1+terminator
 +
 +
|}
 +
&nbsp;
 +
 +
 +
'''Procedure'''
 +
 +
total volume 20 μl
 +
 +
 +
1. add H2O(17 μl -X-Y-Z)
 +
 +
2. add 2 μl Ligase Buffer 10x
 +
 +
3. add Insert 1, Insert 2 (when proceeding from 3A digestion use 2 μl of each)
 +
 +
4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
 +
 +
5. Add 1 μl T4-DNA Ligase
 +
 +
6. Incubate&nbsp; 10-30 min at room temperature
 +
 +
7. heat for 20 minutes at 80°C
 +
 +
8. store at -20°C or directly proceed to transformation
 +
 +
&nbsp;
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Name of part
 +
| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Ratio Insert:Vector
 +
 +
1:1
 +
| style="border:0.0139in solid #808080;padding:0.0194in;"| Volume (μl)
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| X insert 1
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;ho1/ pcyA
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;2
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Y insert 2
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;terminator
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;2
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Z vector
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;pSB1C3
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;2.5
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| H2O
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
 +
| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;11
 +
 +
|}
 +
 +
 +
Competent neb10 strain was transformed with 2μl ligation reaction and plated out.
===PCR===
===PCR===
Line 43: Line 237:
Primer:
Primer:
-
*P 10
+
 
-
*P 11
+
{| style="border-spacing:0;"
 +
| style="border-top:0.0007in solid #000000;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| P10
 +
| style="border:0.0007in solid #000000;padding:0.0382in;"| atatgaattcgcggccgcttctagATGGCCACCACCGTAC
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| P11
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:0.0007in solid #000000;padding:0.0382in;"| atatatgcatctgcagcggccgctactagtaTTACCCTTCTTTTGTCaTGCC
 +
 
 +
|}
 +
 
 +
 
DNA template:
DNA template:
*BBa_I150010
*BBa_I150010
 +
 +
Programm:
 +
2min 55/60
<br/>
<br/>
<br/>
<br/>
Line 144: Line 351:
|}
|}
 +
 +
<br>
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==

Latest revision as of 00:55, 22 September 2011


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

Contents

red light receptor

3A-assembly: Digest

Investigators: Jakob

3A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)


Digestion


Name:Jakob Date: 14.07.2011
Project Name: 3A-assembly of pcyA+ terminator, ho1+terminator

 


Procedure

 

1. add H2O (16μl-DNA )   

2. 2μl NEB4 buffer

3. 2 μl 10x BSA     (used 1:10 diluted )

4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)

5. 1 μl restriction enzymes

6. heat for 1-2 hours 37°C (6 hours if time)

7. heat for 20 minutes 80°C (inactivation of enzymes)

8. keep at 4°C if you cannot continue

 

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:


Sample Name DNA concentration (μg/μl)
Vector  pSB1T3 20
terminator 38.8
ho1 176
pcyA 173

 

Restriction enzymes you need to cut the vector, insert1 and insert 2:


Components
Vector (μl)
Insert1 and 2 (μl)
DNA (μl) 2 Ho1 2.5

pcyA 2.5

Terminator 5.5

terminator 5.5

BSA (10x) (2μl)      
NEB4 Buffer (2μl)      
Enzyme 1 (1μl)  EcoRI  EcoRI  XbaI
Enzyme 2 (1μl)  PstI  SpeI  PstI
H2O (14 μl- DNA)      
In total 20 μl      

 

3A-assembly: Ligation and transformation

Investigators: Julia

Ligation of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator) in vector pSB1T3.

L23: Ligation of S23+S21a in pSB1T3 L22: Ligation of S22+S21a in pSB1T3 Ligation


Name:Julia Date:14.07
Continue from                                    Date14.07 Name

Experiment     3A-assembly

Project Name:3A-assembly of pcyA+ terminator, ho1+terminator

 


Procedure

total volume 20 μl


1. add H2O(17 μl -X-Y-Z)

2. add 2 μl Ligase Buffer 10x

3. add Insert 1, Insert 2 (when proceeding from 3A digestion use 2 μl of each)

4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)

5. Add 1 μl T4-DNA Ligase

6. Incubate  10-30 min at room temperature

7. heat for 20 minutes at 80°C

8. store at -20°C or directly proceed to transformation

 


  Name of part Ratio Insert:Vector

1:1

Volume (μl)
X insert 1  ho1/ pcyA    2
Y insert 2  terminator    2
Z vector  pSB1C3    2.5
H2O      11


Competent neb10 strain was transformed with 2μl ligation reaction and plated out.

PCR

Investigators: Julia

PCR of the red light receptor, cph8.

Primer:

P10 atatgaattcgcggccgcttctagATGGCCACCACCGTAC
P11 atatatgcatctgcagcggccgctactagtaTTACCCTTCTTTTGTCaTGCC


DNA template:

  • BBa_I150010

Programm: 2min 55/60

Lysis cassette

Quickchange PCR


Name: Theo


Date: 14.07.2011
Continue from Experiment Lysis Cassette



Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from

Lysis Device

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 6x HF Phusion Buffer
2.5µl Primer fw P29 (1:10)
2.5µl Primer dw P30 (1:10)
1µl dNTPs
1µl DNA-Template M15a (from S15->diluted to 5ng/µl)
0.5 µl Phusion (add in the end)

What program do you use?


95°C - 15 sec
95°C - 5 min
55°C - 15 sec
72°C -5 min
16°C - ∞
72°C - 5’ + 5’’
20x


Precipitator

Test-digest

Investigators: Rüdiger

Testdigest of the minipreps, Sophie did (june, 13).

  • Enzyme 1: PstI
  • Enzyme 2: EcoRI


caption

Image of different samples from miniprep.

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/14_July"