Team:Stockholm/25 September 2010

From 2010.igem.org

(Difference between revisions)

Latest revision as of 11:06, 26 October 2010

Andreas

Cloning and assembly

Digestion

Further digestions in addition to the ones performed 24/9.

	pK.N-TAT⋅ SH (4) X+P	pK.N-LMWP⋅ SH (1) X+P	pK.N-LMWP⋅ SH (1) S+P
10X FastDigest buffer	2	2	2
DNA (1 μg)	8	4	4
dH₂O	8	12	12
FD XbaI	1	1	0
FD SpeI	0	0	1
FD PstI	1	1	1
	20 μl	20 μl	20 μl

Incubation: 37 °C, 1 h
Inactivation: 80 °C, 30 min (including digestion samples from 24/9)

Ligation

Underlined sequences relevant for assembly

	1	2	3	4	5	6	7	8
10X T4 Ligase buffer	2	2	2	2	2	2	2	2
Vector DNA (50 ng)	1	1	1	1	1	1	1	1
Insert DNA	5	5	5	5	4	4	4	4
dH₂O	11	11	11	11	12	12	12	12
T4 DNA ligase	1	1	1	1	1	1	1	1
	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl

pEX.N-LMWP⋅SOD⋅His (K)
- pSB1K3.N-LMWP⋅SOD⋅His X+P
- pEX.RFP X+P
pEX.N-TAT⋅SOD⋅His
- pSB1K3.N-TAT⋅SOD⋅His X+P
- pEX.RFP X+P
pEX.N-Tra10⋅SOD⋅His
- pSB1K3.N-Tra10⋅SOD⋅His X+P
- pEX.RFP X+P
pEX.N-LMWP⋅SOD⋅His (C)
- pSB1C3.N-LMWP⋅SOD⋅His X+P
- pEX.RFP X+P
pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS
- pSB1K3.N-LMWP⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS
- pSB1K3.N-TAT⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS
- pSB1K3.N-Tra10⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS
- pSB1C3.N-LMWP⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P

Transformation

Standard transformation according to protocol.
- 1 μl ligation mix
- Amp 100, Cm 25 or Km 50 plates
- 50 μl IPTG (pEX constructs, Amp 100 plates)

Sequencing preparation

Prepared samples for sequencing to be sent on Monday.

Construct	Primer	Sample name	Sequencing code (edited 27/9)
pEX.N-TAT⋅SOD⋅His 3	pEXf	pEX.nTAT*SH3_pEXf	ASB0045 614
pEX.N-TAT⋅SOD⋅His 4	pEXf	pEX.nTAT*SH4_pEXf	ASB0045 615
pSB1A2.RBS.yCCS 3	VF2	pA.RBS.yCCS3_VF2	ASB0045 603
pSB1A2.RBS.yCCS 4	VF2	pA.RBS.yCCS4_VF2	ASB0045 604
pSB1K3.N-TAT⋅SOD⋅His 4	VF2	pK.nTAT*SH4_VF2	ASB0045 608
pSB1K3.N-TAT⋅SOD⋅His 5	VF2	pK.nTAT*SH5_VF2	ASB0045 609
pSB1K3.N-Tra10⋅SOD⋅His 5	VF2	pK.nTra10*SH5_VF2	ASB0045 610
pSB1K3.N-LMWP⋅SOD⋅His 1	VF2	pK.nLMWP*SH1_VF2	ASB0045 607
pSB1C3.N-LMWP⋅SOD⋅His 1	VF2	pC.nLMWP*SH1_VF2	ASB0045 605
pSB1C3.N-LMWP⋅SOD⋅His 4	VF2	pC.nLMWP*SH4_VF2	ASB0045 606
pEX.SOD	pEXf	pEX.SOD_pEXf	ASB0045 611
pEX.yCCS 5	pEXf	pEX.yCCS5_pEXf	ASB0045 616
pEX.SOD⋅His	pEXf	pEX.SH_pEXf	ASB0045 612
pEX.His⋅SOD	pEXf	pEX.HS_pEXf	ASB0045 613

BL21 transformation

Transformed BL21 cells with not-yet-verified pEX.N-TAT⋅SOD⋅His plasmid samples 3 & 4

Standard transformation according to protocol.
- 1 μl ligation mix
  - 100 μl cell aliquot divided in 2x50 μl to lower number of colonies after transformation.
- Amp 100 plates

Preparation of LB agar plates

Made new Cm (25 μg/ml) and Km (50 μg/ml) LB agar plates.

10X Cm 25
10X Km 50

The Faculty of Science at Stockholm University

Swedish Vitiligo association (Svenska Vitiligoförbundet)

@@ Line 1: / Line 1: @@
 {{Stockholm/Top2}}
 ==Andreas==
@@ Line 9: / Line 10: @@
 |&nbsp;
 !width="70"|pK.N-TAT&sdot; SH (4) X+P
-!width="70"|pK.N-LMWP&sdot; SH (4) X+P
+!width="70"|pK.N-LMWP&sdot; SH (1) X+P
-!width="70"|pK.N-LMWP&sdot; SH (4) S+P
+!width="70"|pK.N-LMWP&sdot; SH (1) S+P
 |-
 |10X FastDigest buffer
@@ Line 126: / Line 127: @@
 #'''pEX.N-LMWP&sdot;SOD&sdot;His (K)'''
 #*pSB1K3.<u>N-LMWP&sdot;SOD&sdot;His</u> X+P
-#*<u>pEX</u>.SOD X+P
+#*<u>pEX</u>.RFP X+P
 #'''pEX.N-TAT&sdot;SOD&sdot;His'''
 #*pSB1K3.<u>N-TAT&sdot;SOD&sdot;His</u> X+P
-#*<u>pEX</u>.SOD X+P
+#*<u>pEX</u>.RFP X+P
 #'''pEX.N-Tra10&sdot;SOD&sdot;His'''
 #*pSB1K3.<u>N-Tra10&sdot;SOD&sdot;His</u> X+P
-#*<u>pEX</u>.SOD X+P
+#*<u>pEX</u>.RFP X+P
 #'''pEX.N-LMWP&sdot;SOD&sdot;His (C)'''
 #*pSB1C3.<u>N-LMWP&sdot;SOD&sdot;His</u> X+P
-#*<u>pEX</u>.SOD X+P
+#*<u>pEX</u>.RFP X+P
-#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His'''
+#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS'''
 #*<u>pSB1K3.N-LMWP&sdot;SOD&sdot;His</u> S+P
 #*pSB1A2<u>.RBS.yCCS</u> X+P
-#'''pSB1K3.N-TAT&sdot;SOD&sdot;His'''
+#'''pSB1K3.N-TAT&sdot;SOD&sdot;His.RBS.yCCS'''
 #*<u>pSB1K3.N-TAT&sdot;SOD&sdot;His</u> S+P
 #*pSB1A2<u>.RBS.yCCS</u> X+P
-#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His'''
+#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His.RBS.yCCS'''
 #*<u>pSB1K3.N-Tra10&sdot;SOD&sdot;His</u> S+P
 #*pSB1A2<u>.RBS.yCCS</u> X+P
-#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His'''
+#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS'''
 #*<u>pSB1C3.N-LMWP&sdot;SOD&sdot;His</u> S+P
 #*pSB1A2<u>.RBS.yCCS</u> X+P
@@ Line 162: / Line 163: @@
 !Primer
 !Sample name
+!Sequencing code<br />(edited 27/9)
 |-
 |pEX.N-TAT&sdot;SOD&sdot;His 3
 |pEXf
 |pEX.nTAT*SH3_pEXf
+|ASB0045 614
 |-
 |pEX.N-TAT&sdot;SOD&sdot;His 4
 |pEXf
 |pEX.nTAT*SH4_pEXf
+|ASB0045 615
 |-
 |pSB1A2.RBS.yCCS 3
 |VF2
 |pA.RBS.yCCS3_VF2
+|ASB0045 603
 |-
 |pSB1A2.RBS.yCCS 4
 |VF2
 |pA.RBS.yCCS4_VF2
+|ASB0045 604
 |-
 |pSB1K3.N-TAT&sdot;SOD&sdot;His 4
 |VF2
 |pK.nTAT*SH4_VF2
+|ASB0045 608
 |-
 |pSB1K3.N-TAT&sdot;SOD&sdot;His 5
 |VF2
 |pK.nTAT*SH5_VF2
+|ASB0045 609
 |-
 |pSB1K3.N-Tra10&sdot;SOD&sdot;His 5
 |VF2
 |pK.nTra10*SH5_VF2
+|ASB0045 610
 |-
 |pSB1K3.N-LMWP&sdot;SOD&sdot;His 1
 |VF2
 |pK.nLMWP*SH1_VF2
+|ASB0045 607
 |-
 |pSB1C3.N-LMWP&sdot;SOD&sdot;His 1
 |VF2
 |pC.nLMWP*SH1_VF2
+|ASB0045 605
 |-
 |pSB1C3.N-LMWP&sdot;SOD&sdot;His 4
 |VF2
 |pC.nLMWP*SH4_VF2
+|ASB0045 606
 |-
 |pEX.SOD
 |pEXf
 |pEX.SOD_pEXf
+|ASB0045 611
 |-
 |pEX.yCCS 5
 |pEXf
 |pEX.yCCS5_pEXf
+|ASB0045 616
 |-
 |pEX.SOD&sdot;His
 |pEXf
 |pEX.SH_pEXf
+|ASB0045 612
 |-
 |pEX.His&sdot;SOD
 |pEXf
 |pEX.HS_pEXf
+|ASB0045 613
 |}
@@ Line 231: / Line 247: @@
 *10X Cm 25
 *10X Km 50
+{{Stockholm/Footer}}