Team:Freiburg/Notebook/19 July

From 2011.igem.org

(Difference between revisions)
(→NAME OF YOUR EXPERIMENT)
(→Precipitator)
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Lane10
Lane10
60 S14+P20+P28
60 S14+P20+P28
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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: Ruediger
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 19.07
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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;"| Continue from Experiment (Date) 19.07 PCR
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(Name) Ruediger
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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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GFP Pbd
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|}
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Procedure
 +
 +
 +
# add H<sub>2</sub>O (38μl-DNA )
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# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
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# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
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# DNA (500 ng)
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# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
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# heat for 1-2 hours 37°C (6 hours if time)
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# heat for 20 minutes 80°C (inactivation of enzymes)
 +
# keep at 4°C if you cannot continue
 +
 +
Vector (ratio 1:3 to insert)
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 +
Inserts (500ng)
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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;"| '''Components'''
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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;"| Vector (μl)
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| colspan="3"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Insert1 (μl)
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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;"| DNA (500ng)
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5,6
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 12,5
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4,7
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3,5
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3,8
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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;"| BSA (100x) (5μl)
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;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;"|
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;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-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| NEB4 Buffer (5μl)
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;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-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 1 (1μl)
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| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SpeI
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| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SpeI
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| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
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| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
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| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
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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;"| Enzyme 2 (1μl)
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
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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;"| H<sub>2</sub>O (38 μl- DNA)
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 25,5
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 33,7
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 34,5
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 34,2
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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;"| In total 50 μl
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;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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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;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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Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the 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;"| Sample
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA concentration (μg/μl)
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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;"| S14+P20+P28 (short:20)
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 132
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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;"| S14+P19+P28 (short 19)
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| 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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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S14+P18+P28 (short 18)
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| 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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|-
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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
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| 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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|-
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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
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| 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;"|
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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: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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'''Documentation:'''
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 +
Why are you doing this experiment? Where are the samples stored? Name of samples, antibiotica resistance, vector used etc.
 +
 +
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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;"| Want to ligate GFPpbd (3 different versions P18/19/20) into PR vectors (one with strong Promotor strong RBS, one with middle Promotor, middle RBS)
 +
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CM Resistance
 +
 +
Mistake: took 100x BSA instead of 10X
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|}

Revision as of 14:12, 20 July 2011

Contents

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME


blue light receptor

Theoretical Gibson-Assembly

Investigators: Sandra, Sophie

Primer Degin for Blue light sensor (lovTAP) + trp promotor (BBa_K322999) and tetR Gen + tetO promotor (BBa_Q04400).

  • LOVtap-Trp_up: gaattcgcggccgcttctagtcacacaggaaagtactatgt
  • LOVtap-Trp_dw: tacttttatctaatctggacatctagtatttctcctctttgtcgataccctttttacgtg
  • TetR-TetO_up: aaagaggagaaatactagatgtccagattag
  • TetR-TetO_dw^: ctgcagcggccgctactag

^we also have another primer for this one, but we are not sure about the length/melting temperature. Might be changed.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

PCR


Name: Ruediger


Date: 19.07.11
Continue from Experiment (Date)

PCR 18.07 (Name)

Project Name: GFP Pbd

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 P18, P19, P20
2.5µl Primer dw P28
1µl dNTPs of Template DNA
1µl DNA-Template PCR product of P1,P3,S14 from yesterday
0.5 µl Phusion (add in the end)

What program do you use?

One set of probes was prepared and then split into 3 tubes each to test them at Annealingtemperatures 44C, 52C and 60C for the first 10 cycles. Then Annealingtemperature 60C for 25 cycles


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?

S14+P18+P28

S14+P19+P28

S14+P20+P28

Stored in PCR product box

Freiburg11 Phusion ladder.JPG Freiburg11 7 20 2011 2 48 01 PM.Jpg

Lane1 Quick Load Marker Lane2 44C S14+P18+P28 Lane3 44C S14+P19+P28 Lane4 44C S14+P20+P28 Lane5 52C S14+P18+P28 Lane6 52C S14+P19+P28 Lane7 52C S14+P20+P28 Lane8 60 S14+P18+P28 Lane9 60 S14+P19+P28 Lane10 60 S14+P20+P28



Digestion


Name: Ruediger


Date: 19.07
Continue from Experiment (Date) 19.07 PCR

(Name) Ruediger

Project Name:

GFP Pbd

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)
  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

Vector (ratio 1:3 to insert)

Inserts (500ng)


Components Vector (μl) Insert1 (μl)
DNA (500ng) 5,6 12,5 4,7 3,5 3,8
BSA (100x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl) SpeI SpeI XbaI XbaI XbaI
Enzyme 2 (1μl) PstI PstI PstI PstI PstI
H2O (38 μl- DNA) 32,5 25,5 33,7 34,5 34,2
In total 50 μl

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


Sample DNA concentration (μg/μl)
S14+P20+P28 (short:20) 132
S14+P19+P28 (short 19) 145
S14+P18+P28 (short 18) 107
S39 90
S43 40

Documentation:

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


Want to ligate GFPpbd (3 different versions P18/19/20) into PR vectors (one with strong Promotor strong RBS, one with middle Promotor, middle RBS)

CM Resistance

Mistake: took 100x BSA instead of 10X

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/19_July"