Paris/July 24

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[[Paris/July 23|yesterday]] -- [[Paris/July 25|tomorrow]] <br>
==Minipreps==
==Minipreps==
-
<bbpart>BBa_E0422</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - MP6.1' & MP6.2'<br>
+
<bbpart>BBa_E0422</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - [[Paris/Freezer#Plasmids:_MiniPrep_Products|MP6.1' & MP6.2']]<br>
-
<bbpart>BBa_E0241</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1 - MP7.1'<br>
+
<bbpart>BBa_E0241</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1 - [[Paris/Freezer#Plasmids:_MiniPrep_Products|MP7.1']]<br>
-
<bbpart>BBa_J61047</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - MP9.1' & MP9.2'<br>
+
<bbpart>BBa_J61047</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - [[Paris/Freezer#Plasmids:_MiniPrep_Products|MP9.1' & MP9.2']]<br>
 +
 
 +
== Ligation & transformation reactions ==
 +
 
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Ligations
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|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=25%| Insert
 +
|width=5%| Insert Volume
 +
|width=25%| Vector
 +
|width=5%| Vector Volume
 +
|width=35%| Comments
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L1
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|D23 (AraC/pBad promoter FI)
 +
|
 +
|D9 (J61002 ready for insertion of a FI)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L2
 +
|D18 (BB dig. lox66DapAE.coli)
 +
|
 +
|D22 (pSB1A2 Eco, Pst)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L3
 +
|D19 (BB dig. lox66DapAsubtilis)
 +
|
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|D22 (pSB1A2 Eco, Pst)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L4
 +
|D20 (Lox66-DapAE.coli BI)
 +
|
 +
|D1 (pJ23100 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L5
 +
|D21 (lox66-DapAsubtilis BI)
 +
|
 +
|D1 (pJ23100 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L6
 +
|D20 (Lox66-DapAE.coli BI)
 +
|
 +
|D3 (pJ23107 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L7
 +
|D21 (lox66-DapAsubtilis BI)
 +
|
 +
|D3 (pJ23107 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L8
 +
|D14 (Cre ORF)
 +
|
 +
|D15 (B0030 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L9
 +
|D27 (Lox71-ftsZ BI)
 +
|
 +
|D1 (pJ23100 BV)
 +
|
 +
|
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10
 +
|D27 (Lox71-ftsZ BI)
 +
|
 +
|D3 (pJ23107 BV)
 +
|
 +
|
 +
|}
 +
 
 +
*lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)
 +
 
 +
== Annealing of Lox66 and Lox71 ==
 +
 
 +
Lox66 and Lox71 oligos were designed to form a double-stranded DNA. The extremities bear cohesive overhangs corresponding to digestion by EcoRI and SpeI. See oligos 14 + 15 and 16 + 17 [[Paris/Oligos|here]].
 +
 
 +
'''Annealing mix:'''
 +
* 8 μL of each of the concentrated primers
 +
* 4 μL of salt solution (10 mM NaCl)
 +
* 20 μL of water
 +
 
 +
Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.<br>
 +
More information [http://openwetware.org/wiki/Annealing_complementary_primers here]
 +
 
 +
==Purification of PCR Products==
 +
 
 +
* RBS-DapAColi = P8
 +
* RBS-DapASubtilis = P9
 +
* Lox71-FtsA-FtsZ = P10
 +
* DGAT1 = P3
 +
* DGAT2 = P4

Latest revision as of 17:50, 7 October 2007

yesterday -- tomorrow

Contents

Minipreps

BBa_E0422 in pSB1A2 - Clone 1&2 - MP6.1' & MP6.2'
BBa_E0241 in pSB1A2 - Clone 1 - MP7.1'
BBa_J61047 in pSB1A2 - Clone 1&2 - MP9.1' & MP9.2'

Ligation & transformation reactions

Ligations
Number Insert Insert Volume Vector Vector Volume Comments
L1 D23 (AraC/pBad promoter FI) D9 (J61002 ready for insertion of a FI)
L2 D18 (BB dig. lox66DapAE.coli) D22 (pSB1A2 Eco, Pst)
L3 D19 (BB dig. lox66DapAsubtilis) D22 (pSB1A2 Eco, Pst)
L4 D20 (Lox66-DapAE.coli BI) D1 (pJ23100 BV)
L5 D21 (lox66-DapAsubtilis BI) D1 (pJ23100 BV)
L6 D20 (Lox66-DapAE.coli BI) D3 (pJ23107 BV)
L7 D21 (lox66-DapAsubtilis BI) D3 (pJ23107 BV)
L8 D14 (Cre ORF) D15 (B0030 BV)
L9 D27 (Lox71-ftsZ BI) D1 (pJ23100 BV)
L10 D27 (Lox71-ftsZ BI) D3 (pJ23107 BV)
  • lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)

Annealing of Lox66 and Lox71

Lox66 and Lox71 oligos were designed to form a double-stranded DNA. The extremities bear cohesive overhangs corresponding to digestion by EcoRI and SpeI. See oligos 14 + 15 and 16 + 17 here.

Annealing mix:

  • 8 μL of each of the concentrated primers
  • 4 μL of salt solution (10 mM NaCl)
  • 20 μL of water

Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.
More information here

Purification of PCR Products

  • RBS-DapAColi = P8
  • RBS-DapASubtilis = P9
  • Lox71-FtsA-FtsZ = P10
  • DGAT1 = P3
  • DGAT2 = P4
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