BioWire Materials and Methods

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Contents 1 Experiments 2 Ligation 2.1 Cycle One 2.2 Cycle Two 2.3 Cycle Three 2.4 Cycle Four 2.5 Las System 3 Digestion 3.1 Lux System 3.2 Las System

Experiments

Ligation

Cycle One

(6/21 + 6/22)

Lux System	Las System	Rh1 System	Notes
R0063 + I0462 --> Ia R0062 + E0432 --> Ib C0051 + B0015 --> Ic R0040 + F1610 --> J06001 R0040 + E0422 --> J06003 R0065 + B0030 --> Id.0 R0065 + B0031 --> Id.1 R0065 + B0032 --> Id.2 R0065 + B0033 --> Id.3 R0065 + B0034 --> Id.4	R0079 + B003x --> las-Ia.x C0051 + B0015 --> las-Ib I14016 + B0034 --> las-Ic E0032 + B0015 --> las-Id E0032 + B003x --> las-Ie.x		Ia, Ib, Id.0, Id.2, las-Ib, and las-Id all did not work. They have been moved down into Cycle Two and all dependent ligations have been moved appropriately.

Cycle Two

(6/28)

Lux System	Las System	Rh1 System	Notes
R0063 + I0462 --> Ia R0062 + E0432 --> Ib R0065 + B0030 --> Id.0 R0065 + B0032 --> Id.2 B0030 + Ic --> IIa.0 B0031 + Ic --> IIa.1 B0032 + Ic --> IIa.2 B0033 + Ic --> IIa.3 B0034 + Ic --> IIa.4 C0161 + B0015 --> intermediate2 B0030 + R0040 --> intermediate1.0 B0031 + R0040 --> intermediate1.1 B0032 + R0040 --> intermediate1.2 B0033 + R0040 --> intermediate1.3 B0034 + R0040 --> intermediate1.4	las-Ic + las-Ie.x --> las-IIb.x las-Ic + E0032 --> J06106		We missed las-Ib and las-Id. We'll change the ligation in the next step from las-Ic + las-Id to J06106 + B0015 so that we won't fall behind a step. (Both make J06105.) But we'll have to push off a full cycle on the las-Ib.

Cycle Three

(7/1)

Lux System	Las System	Rh1 System	Notes
Ia + R0062 --> F2621 Ia + Ib --> J06000 Id.0 + E0032 --> J06006.0 Id.1 + E0032 --> J06006.1 Id.2 + E0032 --> J06006.2 Id.3 + E0032 --> J06006.3 Id.4 + E0032 --> J06006.4 B0030 + intermediate2 --> F1611.0 B0031 + intermediate2 --> F1611.1 B0032 + intermediate2 --> F1611.2 B0033 + intermediate2 --> F1611.3 B0034 + intermediate2 --> F1611.4 intermediate1.0 + intermediate2 --> J06002.0 intermediate1.1 + intermediate2 --> J06002.1 intermediate1.2 + intermediate2 --> J06002.2 intermediate1.3 + intermediate2 --> J06002.3 intermediate1.4 + intermediate2 --> J06002.4	C0051 + B0015 --> las-Ib J06106 + B0015 --> J06105

Cycle Four

Lux System	Las System	Rh1 System	Notes
F2621 + IIa.0 --> J06004.0 F2621 + IIa.1 --> J06004.1 F2621 + IIa.2 --> J06004.2 F2621 + IIa.3 --> J06004.3 F2621 + IIa.4 --> J06004.4 J06006.0 + B0015 --> J06005.0 J06006.1 + B0015 --> J06005.1 J06006.2 + B0015 --> J06005.2 J06006.3 + B0015 --> J06005.3 J06006.4 + B0015 --> J06005.4 J06006.0 + F1610 --> J06007.0 J06006.1 + F1610 --> J06007.1 J06006.2 + F1610 --> J06007.2 J06006.3 + F1610 --> J06007.3 J06006.4 + F1610 --> J06007.4 J06006.0 + F1611.0 --> J06008.0 J06006.1 + F1611.1 --> J06008.1 J06006.2 + F1611.2 --> J06008.2 J06006.3 + F1611.3 --> J06008.3 J06006.4 + F1611.4 --> J06008.4	las-Ia.x + las-Ib --> las-IIa.x

Las System

Have but not ready: I14018
Have ordered: I0464/P0479, P0478, E0434

General stuff:

• I14018 + I0464 + R0079 -> J06400
• B0034 + C0178 + B0015 -> J06401

• J06400 + E0434 -> J06100
• R0040 + P0478 -> J06101
• R0040 + J06401 -> J06102
• J06003 = J06103
• J06400 + B0030 + C0051 + B0015 -> J06104
• J06106 + B0015 -> J06105
• I14016 + B0034 + E0032 -> J06106
• I14016 + B0030 + E0032 + P0478 -> J06107
• J06106 + J06401 -> J06108

Digestion

Lux System

• Step One

Day One (6/20)		Day Two (6/21)
Vector Insert R0063 I0462 R0062 E0432 C0051 B0015 R0040 F1610 R0040 E0422		Vector Insert B0030 R0065 B0031 R0065 B0032 R0065 B0033 R0065 B0034 R0065

• Step Two (6/24)

Vector Insert Method R0062 Ia prefix B0030 Ic suffix B0031 Ic suffix B0032 Ic suffix B0033 Ic suffix B0034 Ic suffix

Vector Insert Method Ia Ib suffix Id.0 E0032 suffix Id.1 E0032 suffix Id.2 E0032 suffix Id.3 E0032 suffix Id.4 E0032 suffix

Las System

• Step One (6/24)

Vector	Insert	Method
B003X	R0079	prefix
B0015	C0051	prefix
B0034	I14016	prefix
B003X	E0032	prefix
B0015	E0032	prefix

Step Two:

Vector	Insert	Method
las-Ia.x	las-Ib	suffix
las-Ic	las-Id	suffix
las-Ic	las-Ie.x	suffix
las-Ic	E0032	suffix