Paris/July 27

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Latest revision as of 17:51, 7 October 2007

PCR to perform

The following PCR reactions have been performed

P5
P6
P8
P9

Oligo design for Wanner deletion

We were for now on not able to perform the deletion of DapA through transduction. So we will try the deletion through the Wanner method. We will also attempt the deletion of FtsZ.

We will use the protocol of the keio collection: [http://ecoli.naist.jp/gb6/Resources/deletion/Del_construct.html]

20nt of upstream region of kan gene,

5'-ATTCCGGGGATCCGTCGACC-3' (P1)

20nt of kan downstream sequence,

5'-TGTAGGCTGGAGCTGCTTCG-3' (P2)

DapA deletion

50nt DapA upstream region

5'-CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATG-3' (P3)

50nt DapA downstream region (reverse complement)

5'-AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGC-3' (P4)

dDapA-F

CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATGATTCCGGGGATCCGTCGACC (P3 + P1)

dDapA-R

AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGCTGTAGGCTGGAGCTGCTTCG (P6 + P2)

FtsZ deletion

50nt FtsZ upstream region

5'-GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATG-3' (P5)

50nt FtsZ downstream region (reverse complement)

5'-GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGC-3' (P6)

dFtsZ-F

GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATGATTCCGGGGATCCGTCGACC (P5 + P1)

dFtsZ-R

GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGCTGTAGGCTGGAGCTGCTTCG (P6 + P2)

Ligations

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments	Number of colonies
Control 0			pUC19		control of transformation efficiency	4
Control 1			MP3.1 digested with EcoRI (D0)	2	control of ligation efficiency	~80
Control 2			MP3.1 digested with EcoRI (D0)	2	without ligase in the mix: control of digestion efficiency	0
Control 3			D9.1 (J61002 ready for insertion of a FI)	2µl	control of D9.2 digestion	6
Control 4			D15.1 (B0030 BV)	2µl	control of D15.2 digestion	1
Control 5			D22.2 (pSB1A2 Eco, Pst)	2µl	control of D22.1 digestion	0
L1	D23.2 (AraC/pBad promoter FI)	8	D9.1 (J61002 ready for insertion of a FI)	2	construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)	12
L6	D14.2(Cre ORF)	8	D15.1 (B0030 BV)	2	construction of the biobrick strong RBS(B0030)-Cre ORF	~150
L9	lox66 [O14+O15] (0.2µM)	2	D22.2 (pSB1A2 Eco, Pst)	2 (+6 H2O)	lox66 cloned as a biobrick in pSB1A2	0
L10	lox71 [O16+O17] (0.2µM)	2	D22.2 (pSB1A2 Eco, Pst)	2 (+6 H2O)	lox71 cloned as a biobrick	0

Ligation mix:

2µl TP
1µl Ligase
7µl H20

Reaction in 20µl

ON @ 4°C

Minipreps

minipreps were performed on the following ON cultures:

cultures of different clones of the ligation reactions of 25/07/07

L1'.2 & L1'.3
L2'.1 & L2'.2
L9'
L10'
L6'.1, L6'.2 & L6'.3

for more detail on these ligation reactions, go to 25/07/07

no glycerol stocks of hese strains have been generated yet (the culyures are launched tonight in order to do this)

minipreps & glycerol stocks:

MP12.1 & MP12.2 (2 clones after transformation with biobrick BBa_P1003)

@@ Line 1: / Line 1: @@
+[[Paris/July 26|yesterday]] -- [[Paris/July 28|tomorrow]] <br>
 == PCR to perform ==
@@ Line 52: / Line 53: @@
 |- style="background: #ccccff; text-align: center;"
 |width=5%| Number
-|width=25%| Insert
+|width=20%| Insert
 |width=5%| Insert Volume (µL)
 |width=25%| Vector
 |width=5%| Vector Volume (µL)
 |width=35%| Comments
+|width=5%| Number of colonies
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |Control 1
+| style="background: #ccffcc;" |Control 0
 |
 |
-|MP3.1 digested with EcoRI
+|pUC19
 |
+|control of transformation efficiency
+|4
+|- style="background: #cccccc;"
+| style="background: #ccffcc;" |Control 1
+|
+|
+|MP3.1 digested with EcoRI (D0)
+|2
 |control of ligation efficiency
+|~80
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |Control 2
 |
 |
-|MP3.1 digested with EcoRI
+|MP3.1 digested with EcoRI (D0)
-|
+|2
 |without ligase in the mix: control of digestion efficiency
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |Control 3
@@ Line 78: / Line 90: @@
 |2µl
 |control of D9.2 digestion
+|6
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |Control 3
+| style="background: #ccffcc;" |Control 4
 |
 |
@@ Line 85: / Line 98: @@
 |2µl
 |control of D15.2 digestion
+|1
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |Control 3
+| style="background: #ccffcc;" |Control 5
 |
 |
@@ Line 92: / Line 106: @@
 |2µl
 |control of D22.1 digestion
+|0
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |L1
+| style="background: #ccffcc;" |L1''
 |D23.2 (AraC/pBad promoter FI)
 |8
@@ Line 99: / Line 114: @@
 |2
 |construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)
+|12
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |L6
+| style="background: #ccffcc;" |L6''
 |D14.2(Cre ORF)
 |8
@@ Line 106: / Line 122: @@
 |2
 |construction of the biobrick strong RBS(B0030)-Cre ORF
+|~150
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |L9
+| style="background: #ccffcc;" |L9''
 |lox66 [O14+O15] (0.2µM)
 |2
@@ Line 113: / Line 130: @@
 |2 (+6 H2O)
 |lox66 cloned as a biobrick in pSB1A2
+|0
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |L10
+| style="background: #ccffcc;" |L10''
 |lox71 [O16+O17] (0.2µM)
 |2
@@ Line 120: / Line 138: @@
 |2 (+6 H2O)
 |lox71 cloned as a biobrick
+|0
 |}
@@ Line 136: / Line 155: @@
 cultures of different clones of the ligation reactions of 25/07/07
-*L1.2 & L1.3
+*L1'.2 & L1'.3
-*L2.1 & L2.2
+*L2'.1 & L2'.2
-*L9
+*L9'
-*L10
+*L10'
-*L6.1, L6.2 & L6.3
+*L6'.1, L6'.2 & L6'.3
 for more detail on these ligation reactions, go to [[Paris/July 25|25/07/07]]

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