Paris/July 30

From 2007.igem.org

(Difference between revisions)

Revision as of 13:33, 1 August 2007

Sequences

All the biobricks that we got from the plate and all the ligations that succeeded up to now.

Colonies PCR screen

On colony PCRs performed to screen for correct ligation reactions (performed on 27/07/07):

10 clones of L1 ligation reactions, 6 clones of L6 ligation reactions

preparation of the following PCR mix

18x (12.5µL 2xreaction mix)= 225

18x (0.5µL oligo O18 VF2 10µM)=9
18x (0.5µL oligo O19 VR 10µM)=9

12µL H20+colony (10min 100°)

PCRs

PCR : P5 lox66-DapAColi

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		985
55	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	O6	2.5µL
2m00'	Oligo R 10µM	O7	2.5µL	Image (click to enlarge)
Number cycles	Water	34µl		[[Image:\|30px]]
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	Toothpick in MG16655 Glycerol

PCR : P6 Lox66-DapASubtilis

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		979
55	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	O8	2.5µL
2m00'	Oligo R 10µM	O9	2.5µL	Image (click to enlarge)
Number cycles	Water	34µl		[[Image:\|30px]]
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	0.5µl Subtilis DNA

PCR : P7 Lox71-FtsZ

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		1260
55	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	O1	2.5µL
2m00'	Oligo R 10µM	O2	2.5µL	Image (click to enlarge)
Number cycles	Water	34µl		[[Image:\|30px]]
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	Toothpick in MG16655 Glycerol

PCR : P8 RBS-DapAColi

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		952
55	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	O20	2.5µL
2m00'	Oligo R 10µM	O7	2.5µL	Image (click to enlarge)
Number cycles	Water	34µl		[[Image:\|30px]]
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	Toothpick in MG16655 Glycerol

PCR : P11 Lox71-KmR-Lox66

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		1084
55	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	O10	2.5µL
2m00'	Oligo R 10µM	O11	2.5µL	Image (click to enlarge)
Number cycles	Water	34µl		[[Image:\|30px]]
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	0.5 µl MP12

Digestion reactions

The following digestion reactions have been performed:

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	size	Description
D19'	BB dig. lox66DapAsubtilis	lox66-DapAsubtilis P6' PCR product	EcoRI	PstI	~960	PCR product digested for cloning as a BioBrick
D21'	bw-insert lox66-DapAsubtilis	lox66-DapAsubtilis P6' PCR product	XbaI	PstI	~960	PCR product digested for cloning as a BI
D27'	lox71-ftsZ bw-insert	lox71-ftsZ PCR product P7	XbaI	PstI		lox71-ftsZ(unmutated) ready for use as a backward insert
D33	RBS(B0030)CreORF	L6' (clone 1 & 2)	XbaI	PstI		RBS-Cre digestion for use as a BI
D34	lox66	L9' lox66	SpeI	PstI		Biobrick plasmid carrying lox66 digested as a BV
D35	lox71	L10' lox71	SpeI	PstI		Biobrick plasmid carrying lox71 digested as a BV

Ligations

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments	Number of colonies
Control 1			MP3.1 digested with EcoRI (D0)	2	control of ligation efficiency
a faire			MP3.1 digested with EcoRI (D0)	2	without ligase in the mix: control of digestion efficiency
Control 2			D35	2µl	control of D35 digestion	1
Control 3			D34	2µl	control of D34 digestion	2
L3	D19 (lox66dapAsubtilis)	4	D22.1 (pSB1A2 dig. EcoRI&PstI)	2	Insertion of lox66DapASubtilis as a biobrick
L5	D21’(lox66DapASubtilis)	4	D1 (j23100 digested for CDS insertion)	2	construction of the biobrick J23100-lox66dapAsubtilis	0
L7	D27' (lox-ftsZ)	4	D1 (j23100 digested for CDS insertion)	2	construction of the biobrick J23100- lox-ftsZ	0
L8	D27' (lox-ftsZ)	4	D3 (j23107 digested for CDS insertion)	2	construction of the biobrick J23107- lox-ftsZ	0
L9	lox66 [O14+O15] (2µM)	2	D22.2 (pSB1A2 Eco, Pst)	2	lox66 cloned as a biobrick in pSB1A2
L10	lox71 [O16+O17] (2µM)	2	D22.2 (pSB1A2 dig. EcoRI & PstI)	2	lox71 cloned as a biobrick in pSB1A2	0
L11	D21’(lox66dapAsubtilis BI)	4	D3 (j23107 digested for CDS insertion)	2	construction of the biobrick: J23107 promotor-lox66dapAsubtilis	1
L12	D26.1 (gfp-tripart BI)	4	D35 (lox66 plasmid digested as a BV)	2	construction of the biobrick lox66-gfp
L13	D13.2 (mRFP de pJ23100)	4	D34 (lox71 plasmid digested as a BV)	2	lox71 cloned as a biobrick	0
L14	D33.1 (RBS-Cre BI)	4	D1 (j23100 digested for CDS insertion)	2	J23100-RBS-Cre
L15	D33.1 (RBS-Cre BI)	4	D3 (j23107 digested for CDS insertion)	2	J23107-RBS-Cre	0
L16	D32 (lox71-ftsAftsZ BI)	4	D1 (j23100 digested for CDS insertion)	2	J23100-lox71-ftsAftsZ	0
L17	D32 (lox71-ftsAftsZ BI)	4	D3 (j23107 digested for CDS insertion)	2	J23107-lox71-ftsAftsZ	0
L18	D31 (RBSdapAsubtilis BI)	4	D1 (j23100 digested for CDS insertion)	2	J23100-RBSdapAsubtilis	0
L19	D31 (RBSdapAsubtilis BI)	4	D3 (j23107 digested for CDS insertion)	2	J23107-RBSdapAsubtilis	0
L20	D30 (RBSdapAsubtilis dig. BB)	4	D22.2 (pSB1A2 Eco, Pst)	2		0
L21	D29	4	D1 (j23100 digested for CDS insertion)	2		0
L22	D29	4	D3 (j23107 digested for CDS insertion)	2		0
L23	D28 (RBSdapAcoli dig.BB)	4	D22.2 (pSB1A2 Eco, Pst)	2		1
L24	D21’(lox66dapAsubtilis BI)	4	D3 (j23107 digested for CDS insertion)	2		0
L25	D36 (lox71-KmR-lox66 dig. BB)	4	D22.2 (pSB1A2 dig. EcoRI & PstI)	2		1

Ligation mix:

1µl 10x buffer
0.5µl Ligase
2µL plasmid DNA solution
4µL insert DNA solution

Reaction in 10µl

ON @ 4°C

MiniPreps

L1".1, L1".2
L6".1, L6".2

@@ Line 239: / Line 239: @@
 |2µl
 |control of D35 digestion
-|
+|1
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |Control 3
@@ Line 247: / Line 247: @@
 |2µl
 |control of D34 digestion
-|
+|2
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L3''
@@ Line 263: / Line 263: @@
 |2
 |construction of the biobrick J23100-lox66dapAsubtilis
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L7''
@@ Line 271: / Line 271: @@
 |2
 |construction of the biobrick J23100- lox-ftsZ
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L8''
@@ Line 279: / Line 279: @@
 |2
 |construction of the biobrick J23107- lox-ftsZ
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L9''
@@ Line 295: / Line 295: @@
 |2
 |lox71 cloned as a biobrick in pSB1A2
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L11
@@ Line 303: / Line 303: @@
 |2
 |construction of the biobrick: J23107 promotor-lox66dapAsubtilis
-|
+|1
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L12
@@ Line 319: / Line 319: @@
 |2
 |lox71 cloned as a biobrick
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L14
@@ Line 335: / Line 335: @@
 |2
 |J23107-RBS-Cre
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L16
@@ Line 343: / Line 343: @@
 |2
 |J23100-lox71-ftsAftsZ
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L17
@@ Line 351: / Line 351: @@
 |2
 |J23107-lox71-ftsAftsZ
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L18
@@ Line 359: / Line 359: @@
 |2
 |J23100-RBSdapAsubtilis
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L19
@@ Line 367: / Line 367: @@
 |2
 |J23107-RBSdapAsubtilis
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L20
@@ Line 375: / Line 375: @@
 |2
 |
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L21
@@ Line 383: / Line 383: @@
 |2
 |
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L22
@@ Line 391: / Line 391: @@
 |2
 |
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L23
@@ Line 399: / Line 399: @@
 |2
 |
-|
+|1
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L24
@@ Line 407: / Line 407: @@
 |2
 |
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L25
@@ Line 415: / Line 415: @@
 |2
 |
-|
+|1
 |}

Views

Personal tools