Team:Freiburg/Notebook/12 July

From 2011.igem.org

(Difference between revisions)
(→red light receptor)
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===cph8===
===cph8===
S29: Sequence without part. Has to be done again.
S29: Sequence without part. Has to be done again.
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 +
'''Digestion'''
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{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name:
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date:
 +
 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Date Name
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 +
Experiment
 +
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|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
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|}
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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
 +
 +
Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
 +
 +
 +
{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Sample Name
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA concentration (μg/μl)
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P20 GFP
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 132
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P19 GFP
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 145
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P18 GFP
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 107
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S39 PR
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 90
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S43 PR
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 40
 +
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Tet Vector
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 25
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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|}
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{| style="border-spacing:0;"
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| 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;"| '''Components'''
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| 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;"| <center>'''Vector (μl)'''</center>
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| 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;"| <center>'''S39'''</center>
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<center>'''Insert1: PR '''</center>
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| 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;"| <center>'''S43'''</center>
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<center>'''Insert1: PR '''</center>
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| 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;"| <center>'''P18'''</center>
 +
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<center>'''Insert2: GFP+Pbd'''</center>
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| 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;"| <center>'''P19'''</center>
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<center>'''Insert2: GFP+Pbd'''</center>
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| style="border:0.0007in solid #000000;padding:0.0382in;"| <center>'''P20'''</center>
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<center>'''Insert2: GFP+Pbd'''</center>
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| DNA (500ng)
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 20
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''5.5'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''12.5'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''4.7'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''3.5'''</center>
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| 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;"| <center>'''3.8'''</center>
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| BSA (10x) (5μl)
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| 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;"|
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| NEB4 Buffer (5μl)
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| 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;"|
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Enzyme 1 (1μl)
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| E
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''E'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''E'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''X'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''X'''</center>
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| 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;"| <center>'''X'''</center>
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| Enzyme 2 (1μl)
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| P
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''S'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''S'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''P'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''P'''</center>
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| 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;"| <center>'''P'''</center>
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| H<sub>2</sub>O (38 μl- DNA)
 +
| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| 18
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''32.5'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''25.5'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''33.3'''</center>
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| <center>'''34.5'''</center>
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| 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;"| <center>'''34.2'''</center>
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|-
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"| In total 50 μl
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| style="border-top:none;border-bottom:0.0007in solid #000000;border-left:0.0007in solid #000000;border-right:none;padding:0.0382in;"|
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| 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;"|
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|}
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'''Documentation:'''
 +
 +
Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.
 +
 +
 +
{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3A Assembly of linearized empty Tet Vector Psb1T3, PR Parts S39, S43 (CM resistance) and GFP Pbd from PCR with P18,19,20
 +
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I did not digest linearized PsB1T3 with Dpn.
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|}
 +
Run a gel to verify if the part is cut out.
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[[File:12.07.2011_freiburg.jpg]]
==<span style="color:darkblue;">blue light receptor</span>==
==<span style="color:darkblue;">blue light receptor</span>==

Revision as of 13:18, 2 August 2011


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

Contents

Sequence Analysis

Lysis cassette

Lysis cassette and heat inducible promotor

S15b: (Lysis cassette K124014) is named K124017 in the registry Part without RBS at the beginning Part without antiholin the sequece is also mutated leading to wrong amino acids if combined with RBS there would be too many bases (8) between RBS and coding sequence part is likely to be not effective

green light receptor

CcaR

S17: (CcaR with Prefix and RBS) is ok. Check RBS. S18: seems to be ok too.

red light receptor

pycA+terminator

S24: prefix and suffix not complete

cph8

S29: Sequence without part. Has to be done again.

Digestion


Name: Date:
Continue from Date Name

Experiment

Project Name:

Procedure


  1. add H2O (38μl-DNA )
  2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
  3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
  4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
  5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
  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)
P20 GFP 132
P19 GFP 145
P18 GFP 107
S39 PR 90
S43 PR 40
Tet Vector 25
Components
Vector (μl)
S39
Insert1: PR
S43
Insert1: PR
P18
Insert2: GFP+Pbd
P19
Insert2: GFP+Pbd
P20
Insert2: GFP+Pbd
DNA (500ng) 20
5.5
12.5
4.7
3.5
3.8
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl) E
E
E
X
X
X
Enzyme 2 (1μl) P
S
S
P
P
P
H2O (38 μl- DNA) 18
32.5
25.5
33.3
34.5
34.2
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.


3A Assembly of linearized empty Tet Vector Psb1T3, PR Parts S39, S43 (CM resistance) and GFP Pbd from PCR with P18,19,20

I did not digest linearized PsB1T3 with Dpn.

Run a gel to verify if the part is cut out. 12.07.2011 freiburg.jpg

blue light receptor

LOV1

S33: LOVtap only

LOV3

S35: is K322999. This sequence is the LOVtap with the tryptophane promoter, RBS, mRFP and two terminators.

Experiments

green light receptor

Gel

Investigators: Jakob


Comments: Miniprep from June,12 were loaded onto a gel.

Ccas (M31, M32, M30)in pSB1C3

caption

Image of the gel of M30-MM32 and M36-M38

Comments: All the samples were negative.

red light receptor

Gel

Investigators: Jakob


Comments: Miniprep from June,12 were loaded onto a gel. If they are positive: Sequencing

ho1+terminator (M36, M37, M38) in pSB1C3

Comments: All the samples were negative. See image above.

Precipitator

Plastic binding site

Investigators: Sophie

Picking colonies (M2a, M2b, M3), inoculating LB (Cm) and let grow overnight. Start: 17:15

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/12_July"