Paris/September 14

From 2007.igem.org

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[[Paris/September 13|yesterday]] -- [[Paris/September 15|tomorrow]] <br>
 +
== DapA Wanner deletion: pKD3/pKD4-DapA homologies PCR ==
 +
 +
* Material: <br>
 +
 +
- pKD3/pKD4: 1 µL (1.5ng/3ng) <br>
 +
- Tampon GC: 10µL <br>
 +
- dNTP: 1µL (final 200µmol each)<br>
 +
- Taq: 0.5µL<br>
 +
- Water: 32.5µL<br>
 +
- Primer1: 33: 2.5µL (final 0.5µM) <br>
 +
- Primer2: 34: 2.5µL (final 0.5µM)<br>
 +
 +
[[Image:WannerDapA09142007.jpg|right|300px]]
 +
 +
'''FAILED'''
 +
 +
* PCR Program:<br>
 +
 +
35 cycles:<br>
 +
- 98°C: 5min<br>
 +
- 98°C: 30s (X35)<br>
 +
- 69°C: 30s (X35)<br>
 +
- 72°C: 1min30 (X35)<br>
 +
- 72°C: 10 min<br>
 +
- 4°C: inf
 +
 +
* '''Quantity:''' ng/µL (pKD3) et ng/µL (pKD4) <br>
 +
* '''Expected size:'''  1.1 kb (pKD3) / 1.6 kb (pKD4)
 +
 +
PCRs have failed for 4 days because the primers are designed for pKD13.
 +
== Digestions ==
== Digestions ==
Line 16: Line 48:
|width=1%|  
|width=1%|  
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #FF99FF;" |D56
+
| style="background: #ccffcc;" |D56
|araC/pBad>>RBS-Cre BBinsert
|araC/pBad>>RBS-Cre BBinsert
|L26.1 araC/pbad>>RBS-Cre in <bbpart>J61002</bbpart>
|L26.1 araC/pbad>>RBS-Cre in <bbpart>J61002</bbpart>
Line 29: Line 61:
| style="background: #ccffcc;" |D57
| style="background: #ccffcc;" |D57
|lox71-B0015(T) BI
|lox71-B0015(T) BI
-
|L31.1, L31.3, L31.4 lox71-B0015(T) <bbpart>pSB1A2</bbpart>
+
|(L31.1, L31.3) lox71-B0015(T) <bbpart>pSB1A2</bbpart>
|XbaI
|XbaI
|Pst1
|Pst1
Line 40: Line 72:
| style="background: #FF99FF;" |D58
| style="background: #FF99FF;" |D58
|B0015(T)-lox66 BV
|B0015(T)-lox66 BV
-
|L32.1, L32.2, L32.3 B0015(T)-lox66 BV
+
|(L32.1, L32.2) B0015(T)-lox66 BV
|SpeI
|SpeI
|PstI
|PstI
Line 51: Line 83:
| style="background: #ccffcc;" |D59
| style="background: #ccffcc;" |D59
|pTet>>lox71-gfptripart BI
|pTet>>lox71-gfptripart BI
-
|L34.1, L34.2, L34.3 pTet>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
+
|(L34.1, L34.2) pTet>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
|XbaI
|XbaI
|PstI
|PstI
Line 62: Line 94:
| style="background: #ccffcc;" |D60
| style="background: #ccffcc;" |D60
|araC/pBad>>lox71-gfptripart BI
|araC/pBad>>lox71-gfptripart BI
-
|L35.1, L35.2 & L35.3 araC/pBad>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
+
|(L35.1, L35.2) araC/pBad>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
|XbaI
|XbaI
|PstI
|PstI
Line 73: Line 105:
| style="background: #FF99FF;"  |D61
| style="background: #FF99FF;"  |D61
|lox66-mRFP FV
|lox66-mRFP FV
-
|L36.1, L36.2 & L36.3 lox66-mRFP
+
|(L36.1, L36.2) lox66-mRFP
|EcoRI
|EcoRI
|XbaI
|XbaI
|
|
-
|lox66-gfptri part (strongRBS-ORF-T) ready for use as a forward vector
+
|lox66-mRFP part (strongRBS-ORF-T) ready for use as a forward vector
|
|
|
|
Line 84: Line 116:
| style="background: #ccffcc;" |D62
| style="background: #ccffcc;" |D62
|lox66-RBSDapAcoli BI
|lox66-RBSDapAcoli BI
-
|L37.1, L37.2 & L37.3 lox66-RBSDapAcoli  
+
|(L37.1, L37.2) lox66-RBSDapAcoli  
|XbaI
|XbaI
|PstI
|PstI
Line 95: Line 127:
| style="background: #ccffcc;" |D63
| style="background: #ccffcc;" |D63
|RBSDapAcoli BI
|RBSDapAcoli BI
-
|L39.1, L39.2 & L39.3 RBSDapAcoli  
+
|(L39.1, L39.2) RBSDapAcoli  
|XbaI
|XbaI
|PstI
|PstI
Line 106: Line 138:
| style="background: #FF99FF;" |D64
| style="background: #FF99FF;" |D64
|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> BV
|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> BV
-
|MP14.1 <bbpart>BBa_J61025</bbpart>
+
|(MP14.1) <bbpart>BBa_J61025</bbpart>
|SpeI
|SpeI
|PstI
|PstI
Line 117: Line 149:
| style="background: #FF99FF;" |D65
| style="background: #FF99FF;" |D65
|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> FV
|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> FV
-
|MP14.1 <bbpart>BBa_J61025</bbpart>
+
|(MP14.1) <bbpart>BBa_J61025</bbpart>
|EcoR
|EcoR
|XbaI
|XbaI
Line 126: Line 158:
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #FF99FF;" |D65
+
| style="background: #FF99FF;" |D66
|KanR <bbpart>BBa_J61025</bbpart> FV
|KanR <bbpart>BBa_J61025</bbpart> FV
-
|MP12.1 <bbpart>BBa_P1003</bbpart> in <bbpart>pSB1A2</bbpart>
+
|(MP12.1) <bbpart>BBa_P1003</bbpart> in <bbpart>pSB1A2</bbpart>
|EcoR
|EcoR
|XbaI
|XbaI
Line 137: Line 169:
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |D66
+
| style="background: #ccffcc;" |D67
|RBS-DapAsubtilis BI
|RBS-DapAsubtilis BI
-
|L43.1, L43.2 & L43.3 RBS-DapAsubtilis <bbpart>pSB1A2</bbpart>
+
|(L43.1, L43.2) RBS-DapAsubtilis <bbpart>pSB1A2</bbpart>
|XbaI
|XbaI
|PstI
|PstI
Line 148: Line 180:
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #FF99FF;" |D67
+
| style="background: #FF99FF;" |D68
|attB BV
|attB BV
-
|MP15.1
+
|(MP15.1)
|SpeI
|SpeI
|PstI
|PstI
Line 159: Line 191:
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #FF99FF;" |D68
+
| style="background: #FF99FF;" |D69
|gfptripart-lox66 BV
|gfptripart-lox66 BV
-
|L29.1 gfptripart-lox66 <bbpart>pSB1A2</bbpart>
+
|(L29.1) gfptripart-lox66 <bbpart>pSB1A2</bbpart>
|SpeI
|SpeI
|PstI
|PstI
|
|
|
|
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D70
 +
|pTet>>lox71-gfptripart FI
 +
|(L34.1, L34.2) pTet>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
 +
|EcoRI
 +
|SpeI
 +
|
 +
|pTet>>lox71-gfptripart for use as a FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D71
 +
|araC/pBad>>lox71-gfptripart FI
 +
|(L35.1, L35.2) araC/pBad>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
 +
|EcoRI
 +
|SpeI
 +
|
 +
|araC/pBad>>lox71-gfptripart for use as a FI
|
|
|
|
|
|
|}
|}
-
 
-
digestion mix:
 
-
* 25µl plasmid
 
-
* 1µl BSA
 
-
* 10 µl NEB2
 
-
* 60µl H2O
 
-
* 2µl Enzyme1
 
-
* 2µl Enzyme2
 

Latest revision as of 18:04, 7 October 2007

yesterday -- tomorrow

DapA Wanner deletion: pKD3/pKD4-DapA homologies PCR

  • Material:

- pKD3/pKD4: 1 µL (1.5ng/3ng)
- 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:WannerDapA09142007.jpg

FAILED

  • PCR Program:

35 cycles:
- 98°C: 5min
- 98°C: 30s (X35)
- 69°C: 30s (X35)
- 72°C: 1min30 (X35)
- 72°C: 10 min
- 4°C: inf

  • Quantity: ng/µL (pKD3) et ng/µL (pKD4)
  • Expected size: 1.1 kb (pKD3) / 1.6 kb (pKD4)

PCRs have failed for 4 days because the primers are designed for pKD13.

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)
D57 lox71-B0015(T) BI (L31.1, L31.3) lox71-B0015(T) pSB1A2 XbaI Pst1 lox71-B0015(T) extracted for use as a BI pSB1A2
D58 B0015(T)-lox66 BV (L32.1, L32.2) B0015(T)-lox66 BV SpeI PstI B0015(T)-lox66 for use as a BV pSB1A2
D59 pTet>>lox71-gfptripart BI (L34.1, L34.2) pTet>>lox71-gfptripart BBa_J61002 XbaI PstI pTet>>lox71-gfptripart for use as a BI
D60 araC/pBad>>lox71-gfptripart BI (L35.1, L35.2) araC/pBad>>lox71-gfptripart BBa_J61002 XbaI PstI araC/pBad>>lox71-gfptripart for use as a BI
D61 lox66-mRFP FV (L36.1, L36.2) lox66-mRFP EcoRI XbaI lox66-mRFP part (strongRBS-ORF-T) ready for use as a forward vector
D62 lox66-RBSDapAcoli BI (L37.1, L37.2) lox66-RBSDapAcoli XbaI PstI lox66-RBSDapAcoli ready for use as a Backward insert
D63 RBSDapAcoli BI (L39.1, L39.2) RBSDapAcoli XbaI PstI RBSDapAcoli BI (for insertion behind T-lox66)
D64 FRT-CmR-FRT BBa_J61025 BV (MP14.1) BBa_J61025 SpeI PstI FRT-CmR-FRT BBa_J61025 (for backward introduction of a casette for Wanner K insertion)
D65 FRT-CmR-FRT BBa_J61025 FV (MP14.1) BBa_J61025 EcoR XbaI FRT-CmR-FRT BBa_J61025 (for forward introduction of a casette for Wanner K insertion)
D66 KanR BBa_J61025 FV (MP12.1) BBa_P1003 in pSB1A2 EcoR XbaI KanR BBa_J61025 (for forward introduction of a casette for Wanner K insertion)
D67 RBS-DapAsubtilis BI (L43.1, L43.2) RBS-DapAsubtilis pSB1A2 XbaI PstI RBS-DapAsubtilis for use as a BI
D68 attB BV (MP15.1) SpeI PstI vector containing attB for use as a BV
D69 gfptripart-lox66 BV (L29.1) gfptripart-lox66 pSB1A2 SpeI PstI
D70 pTet>>lox71-gfptripart FI (L34.1, L34.2) pTet>>lox71-gfptripart BBa_J61002 EcoRI SpeI pTet>>lox71-gfptripart for use as a FI
D71 araC/pBad>>lox71-gfptripart FI (L35.1, L35.2) araC/pBad>>lox71-gfptripart BBa_J61002 EcoRI SpeI araC/pBad>>lox71-gfptripart for use as a FI
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