Team:Freiburg/Notebook/26 July

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(Sequencing of Quickchanged Lysis Repressor Part (modified K098995))
 
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 26 July </a>
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==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
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===Sequencing of Quickchanged Lysis Repressor Part (modified K098995)===
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===Sequencing of Quickchange-modified Lysis Repressor Part (modified K098995)===
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https://static.igem.org/mediawiki/2011/3/36/Freiburg11_seq_qcLysepromotor_2607.gb
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[https://static.igem.org/mediawiki/2011/3/36/Freiburg11_seq_qcLysepromotor_2607.gb Right-click to download the annotated ape (.gb) file]
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<br>
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===3A Assembly (K098995 + K124017)===
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'''Digestion of 3A Assembly'''
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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: Theo
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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: 26.07.2011
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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 : Quickchange V.3
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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: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)
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|}
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Procedure
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# 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) (Vector:Insert ratio 1:3 in following Ligation)
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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)
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# keep at 4°C if you cannot continue
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Restriction enzymes you need to cut the vector, insert1 and insert 2:
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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;"| <center>'''Vector (μl)'''</center>
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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;"| <center>'''Insert1 -'''K098995-''' and 2 -''' K124017'''-(μl)'''</center>
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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;"| DNA (500ng)
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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;"| 10
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 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;"| 7
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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;"| BSA (10x) (5μl)
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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;"|
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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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| 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)
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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;"|
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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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| 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)
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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;"| EcoRI + DpnI
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| EcoRI
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| style="border: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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| 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)
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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;"| 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;"| SpeI
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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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| 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)
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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;"| 27
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 33
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 31
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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;"| In total 50 μl
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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;"|
 +
| 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? 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;"| 3A assembly of quickchange-modified K098995 with Lysis genes K124017.
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Quickchange-modified K098995: 132,8ng/µl (Amp)
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Lysis genes K124017: (Amp+Kan)
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<nowiki>*Parts were run on the gel and verified that the inserts were cut, image accidentaly</nowiki> not saved*
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Vector: pSB1T3 + DpnI Digestion
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Stored in Theos Box
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|}
<br>
<br>
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<br/>
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'''Ligation'''
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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: Theo
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 26.07.2011
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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: 26.07.2011 Lysis Cassette V.2 Digestion
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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: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)
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|}
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'''Procedure'''
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PCR tube:
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total volume 20 μl
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# add H<sub>2</sub>O (17 μl -X-Y-Z)
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# add 2 μl Ligase Buffer 10x
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# add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
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# add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
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# Add 1 μl T4-DNA Ligase
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# Incubate 10-30 min at room temperature
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# heat for 20 minutes at 80°C
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# store at -20°C or directly proceed to transformation
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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;"|
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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 of part
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ratio Insert:Vector
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<nowiki>= 3:1 or 1:1</nowiki>
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Volume (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;"| X insert 1
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Modif. K098995
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3:1 (1,5*3)
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4,5
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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;"| Y insert 2
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| K124017
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3:1 (2*3)
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 6
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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;"| Z 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;"| pSB1T3
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1:3 (1)
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1
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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
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| style="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="background-color:#bfbfbf;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;"| 5,5
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|}
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'''Documentation:'''
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Why are you doing this experiment? Where are your parts stored? Name the parts for ligation 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;"| Ligation step of 3A assembly.
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How to calculate ratios --> e.g. for K124017
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Length of pSB1T3= ca 2200bp
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Length of K124017+Vector<nowiki>= ca 4500bp</nowiki>
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4500/2200= ca 2
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So 2*3 µl (since 3:1 is needed) = 6 µl
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|}
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<br>
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<br/>
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'''Transformation'''
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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: Theo
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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:
 +
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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 : 26.07.2011 Lysis Cassette V.2 Ligation
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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: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)
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|}
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Procedure
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# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
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# thaw cells on ice 20 minutes
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# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
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# Incubate for 30 minutes on ice
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# Heat at 42°C for 60 sec
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# Incubate on ice for 5 minutes
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# Add 200 μl LB Broth
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# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
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# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
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'''Documentation:'''
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Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance 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;"| New Composite Part in pSB1T3 Vector with quickchange-modified K098995 and Lysis genes K124017
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|}
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<br>
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<br/>

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!