Paris/September 17

From 2007.igem.org

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(Digestions)
 
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[[Paris/September 16|yesterday]] -- [[Paris/September 18|tomorrow]] <br>
== DapA Wanner deletion: pKD13 - DapA homologies PCR ==
== DapA Wanner deletion: pKD13 - DapA homologies PCR ==
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #FF99FF;" |D73
| style="background: #FF99FF;" |D73
-
|<bbpart>pSB2K3</bbpart> BBvector
+
|<bbpart>pSB2K3</bbpart> ES open vector
|MP16 <bbpart>pSB2K3</bbpart>::attP
|MP16 <bbpart>pSB2K3</bbpart>::attP
|EcoRI
|EcoRI

Latest revision as of 18:05, 7 October 2007

yesterday -- tomorrow

DapA Wanner deletion: pKD13 - DapA homologies PCR

  • Material:

- pKD13: 5 µL (bacteria)
- Tampon GC: 10µL
- dNTP: 1µL (final 200µmol each)
- Taq: 0.5µL
- Water: 32.5µL
- Primer1: 33: 2.5µL (final 0.5µM)
- Primer2: 34: 2.5µL (final 0.5µM)

File:WannerDapA09172007.jpg

  • PCR Program:

35 cycles:
- 98°C: 5min
- 98°C: 45s (X35)
- 69°C: 30s (X35)
- 72°C: 1min (X35)
- 72°C: 10 min
- 4°C: inf

  • Quantity: ng/µL
  • Expected size: 1403 kb

Digestions

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D56 araC/pBad>>RBS-Cre BBinsert L26.1 araC/pbad>>RBS-Cre in J61002 EcoRI Pst1 araC/pBad>>RBS-Cre BBinsert (for transfer to the low-copy plasmid vector)
D66 KanR BBa_J61025 FV (MP12.1, MP12.2) BBa_P1003 in pSB1A2 EcoR XbaI KanR BBa_J61025 (for forward introduction of a casette for Wanner K insertion)
D71 araC/pBad>>lox71-gfptripart FI L35.1 araC/pBad>>lox71-gfptripart BBa_J61002 EcoRI SpeI araC/pBad>>lox71-gfptripart for use as a FI
D72 pSB2K3 BBvector MP16 pSB2K3::attP EcoRI PstI Low copy number plasmid, BB vector
D73 pSB2K3 ES open vector MP16 pSB2K3::attP EcoRI SpeI Comparison of digestion efficiency between Pst and Spe in NEB2
D75 AraC-pBAD-RBS-CRE FI L26.1 AraC-pBAD-RBS-CRE EcoRI SpeI FI, Comparison of digestion efficiency between Pst and Spe in NEB2 (see D56)
D76 DGAT BB P14 DGAT EcoRI PstI DGAT