Team:Freiburg/Notebook/26 July

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



Contents

blue light receptor

Gibson-Assembly

Investigators: Sandra, Sophie

Planned Gibson-Assembly for Lov-tap and Not-Gate. Need to order NAD. We will start an thursday.


Precipitator

Ligation


Name: Ruediger Date: 26.07
Continue from Date Name

Experiment Digestion 25.07 Ruediger

Project Name:GFPPbd

Procedure


PCR tube:

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


Part name Volume (μl) Part name Volume (μl)
S39 2 S43 2
P18 1,8 P18 1,8
Psb1T3 2 Psb1T3 2
S39 2 S43 2
P19 1,8 P19 1,8
Psb1T3 2 Psb1T3 2
S39 2 S43 2
P20 1,8 P20 1,8
Psb1T3 2 Psb1T3 2

Added 12,2 μl to every sample


Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.


See digestion yesterday

3A assembly combination as shown in table.


Transformation


Name: RT Date: 26.07
Continue from Date Name

Experiment Ligation 26.07 Ruediger

Project Name: GFPPbd

Procedure


  1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
  2. thaw cells on ice 20 minutes
  3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
  4. Incubate for 30 minutes on ice
  5. Heat at 42°C for 60 sec
  6. Incubate on ice for 5 minutes
  7. Add 200 μl LB Broth
  8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
  9. 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.


Had only 6 tet plates -> put 100 μl of each sample on each plate

Incubator overnight for 30°C


S39-P18

S39-P19

S39-P20


S43-P18

S43-P19

S43-P20


Lysis cassette

Sequencing of Quickchanged Lysis Repressor Part (modified K098995)

>8246585.seq - ID: Lys 55+2 e-P8_25072011 on 2011/7/26-7:48:44 automatically edited with PhredPhrap, start with base no.: 29 Internal Params: Windowsize: 20, Goodqual: 19, Badqual: 10, Minseqlength: 50, nbadelimit: 1


cgaCTTactaTAAatAGGCgtATCACGAGGCAGAATTTCAGATaAAAAAAATCCTTAGCTTTCGCTAAGGaTGATTTCTGGAATTCGCGGCCGCTT
CTAGAGTTTATGGCTAGCTCAGTCCTAGGTACAATGctAGCTACTAGAGAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAA
CACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGAC
AAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCT
CAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTG
AGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTA
AGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCC
TGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGgGGGTGATGAGTTTACCTTCA
AGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTG
GGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGACTCCAGCGGCCGCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC
CTTTCTGCGTTTATATACTAGAGAGAGAATATAAnAAGCCAGATTATTAATCCGGCTTTtTTATTATTTTACTAGAGTAACACCGTGCGTGT
TGACTATTTTACCTCTGGCGGTGataATGGTTGCTACTAGTAGCGGCCGCTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC
GGCTGCGgCgAGcGGTATCAgcTCacTCAAAGgnngGTAATACGGTtaTCcacagaATCAGg





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