Team:Freiburg/Notebook/1 August

From 2011.igem.org

(Difference between revisions)
(3A-assembly with pcyA and the terminator BBa_1006)
 
(17 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:Freiburg/Templates/header}}
{{: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/29_July">Previous entry</a>
 +
</div>
 +
<div id="notebook-title">
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 1 August </a>
 +
</div>
 +
<div id="notebook-next">
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook/2_August">Next entry</a>
 +
</div>
 +
</div>
 +
</html>
 +
==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
Line 11: Line 25:
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===PCR===
 +
 
 +
'''Investigators: Sophie'''
 +
 
-
'''Investigators:NAME''': Sophie
 
-
'''PCR'''
 
As our last PCRs of the LovTAP with the Gibson overhangs didn't work well, we now try a PCR with primers without overhangs for Gibson assembly.
As our last PCRs of the LovTAP with the Gibson overhangs didn't work well, we now try a PCR with primers without overhangs for Gibson assembly.
Line 82: Line 97:
'''To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.'''
'''To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.'''
 +
[[File:Freiburg_2011_08_01.jpg|caption|border]]
'''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? '''
Line 104: Line 120:
Incubated at room temperature for 35 min, inactivated at 80°C for 20 min.<br/>
Incubated at room temperature for 35 min, inactivated at 80°C for 20 min.<br/>
====3.Transformation====
====3.Transformation====
 +
 +
<br>
 +
<br/>
==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Phage Lysis Cassette (K124017) + RBS (B0034)===
-
'''Investigators:NAME'''
+
'''Investigators: Theo'''
 +
<br>
 +
===='''Digestion of 3A Assembly'''====
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 01.08.2011
 +
 +
|-
 +
| 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 : -
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Phage Lysis Cassette (K124017) + RBS (B0034)
 +
 +
|}
 +
Procedure
 +
 +
 +
# add H<sub>2</sub>O (38μl-DNA )
 +
# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
 +
# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
 +
# DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
 +
# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
 +
# heat for 1-2 hours 37°C (6 hours if time)
 +
# heat for 20 minutes 80°C (inactivation of enzymes)
 +
# keep at 4°C if you cannot continue
 +
 +
Restriction enzymes you need to cut the vector, insert1 and insert 2:
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Components'''
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''Vector (μl)'''</center>
 +
| colspan="3"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''Insert1 -'''B0034-''' and 2 -'''K124017'''-(μl)'''</center>
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA (500ng)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 9
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 8
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x) (5μl)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| NEB4 Buffer (5μl)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 1 (1μl)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| EcoRI + DpnI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| EcoRI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 2 (1μl)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SpeI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O (38 μl- DNA)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 27
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 29
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 30
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| In total 50 μl
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|}
 +
'''Documentation:'''
 +
 +
Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| A RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017 is absent. So a 3A assembly had to be performed in order to get a functioning Lysis cassette.
 +
 +
Resistance of RBS (B0034)=Cm and of Phage Lysis Cassette (K124017)=Amp, so the Vector is Tet (pSB1T3)
 +
 +
|}
 +
 +
<br>
 +
<br/>
 +
 +
===='''Ligation'''====
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 01.08.2011
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from: 01.08.2011 Phage Lysis Cassette (K124017) + RBS (B0034) Digestion
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Phage Lysis Cassette (K124017) + RBS (B0034) Ligation
 +
 +
|}
 +
'''Procedure'''
 +
 +
 +
PCR tube:
 +
 +
total volume 20 μl
 +
 +
 +
# add H<sub>2</sub>O (17 μl -X-Y-Z)
 +
# add 2 μl Ligase Buffer 10x
 +
# add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
 +
# add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
 +
# Add 1 μl T4-DNA Ligase
 +
# Incubate 10-30 min at room temperature
 +
# heat for 20 minutes at 80°C
 +
# store at -20°C or directly proceed to transformation
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name of part
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ratio Insert:Vector
 +
 +
<nowiki>= 3:1 or 1:1</nowiki>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Volume (μl)
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| X insert 1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| B0034
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3:1 (1*3)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Y insert 2
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| K124017
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3:1 (2*3)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 6
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Z vector
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| pSB1T3
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1:3 (1)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O
 +
| style="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 7
 +
 +
|}
 +
'''Documentation:'''
 +
 +
Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ligation step of 3A assembly.
 +
 +
 +
How to calculate ratios --> e.g. for K124017
 +
 +
Length of pSB1T3= ca 2200bp
 +
 +
Length of K124017+Vector<nowiki>= ca 4500bp</nowiki>
 +
 +
4500/2200= ca 2
 +
 +
So 2*3 µl (since 3:1 is needed) = 6 µl
 +
 +
 +
 +
 +
|}
 +
 +
<br>
 +
<br/>
 +
 +
===='''Transformation'''====
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 01.08.2011
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from : 01.08.2011 Phage Lysis Cassette (K124017) + RBS (B0034) Ligation
 +
 +
 +
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Phage Lysis Cassette (K124017) + RBS (B0034)
 +
 +
|}
 +
Procedure
 +
 +
 +
# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
 +
# thaw cells on ice 20 minutes
 +
# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
 +
# Incubate for 30 minutes on ice
 +
# Heat at 42°C for 60 sec
 +
# Incubate on ice for 5 minutes
 +
# Add 200 μl LB Broth
 +
# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
 +
# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
 +
 +
'''Documentation:'''
 +
 +
Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| New Composite Part in pSB1T3 Vector with modified K124017 containing a strong RBS in front of first ATG
 +
 +
RESULT: No colonies!!! :( Probably because of the Tet vector, since other members of the group also had problems. We are trying it again with the pSB1C3 Vector
 +
|}
 +
 +
<br>
 +
<br/>
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
-
'''PCR'''
+
===PCR===
Line 180: Line 427:
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
-
 
+
[[File:Freiburg_1.8.11.jpg|capture|border]]
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?
 +
 +
 +
===3A-assembly===
 +
====1.Digestion====
 +
 +
'''Investigators: Theo'''
 +
 +
Digestion of PCR product.
 +
 +
*S39
 +
*S43
 +
 +
were digested with EcoRI and SpeI
 +
 +
*P18
 +
*P19
 +
*P20
 +
 +
were digested with XbaI and PstI.
 +
 +
 +
====2.Ligation====
 +
 +
'''Investigators: Theo'''
 +
 +
Ligation of:
 +
 +
*S39+P18
 +
*S43+P18
 +
*S39+P19
 +
*S43+P19
 +
*S39+P20
 +
*S43+P20
 +
 +
stored in Theo´s box.

Latest revision as of 01:03, 22 September 2011


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