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From 2013.igem.org

Contents 1 RFC10 Standardization of Tyc Modules 1.1 Tyc C5 1.2 Tyc C6 1.3 Tyc A 2013-08-20 1.3.1 Optimiziation A 1.4 Tyc B1 2013-08-21 1.5 Tyc C5 2013-08-21 1.6 Tyc C6 2013-08-21 1.7 Tyc B1 2013-08-22 1.8 Tyc C5 2013-08-22 1.9 Tyc B1 2013-08-23 1.9.1 B 23-08-2013 1.10 Tyc C5 2013-08-23 1.10.1 B 23-08-2013

RFC10 Standardization of Tyc Modules

Tyc C5

what	µl
Bacillus	1
AT05	2
AT06	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [s]
1	98	2:00
35	98	10
	61	0:30
	72	1:00
1	72	2:00
1	10	inf

Tyc C6

what	µl
Bacillus	1
AT07	2
AT08	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [s]
1	98	2:00
35	98	10
	56	0:30
	72	1:00
1	72	2:00
1	10	inf

Tyc A 2013-08-20

Since neither Tyc B1, C5, C6 where succesfully amplified and Tyc A was rather a light band we decided to first find the optimal protocol for Tyc A. Also the Q5 Polymerase was contaminated we decided to use Phusion Flash which is faster and cheaper than the Q5.

Optimiziation A

Reaction

what	µl
Bacillus	1
AT01	2
AT02	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	5

Condition I

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	56	0:05
	72	0:55
1	72	5:00
1	10	inf

Conditions II

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
14	98	0:01
	59 ↓0.5	0:05
	72	0:55
16	98	0:01
	56	0:05
	72	0:55
1	72	5:00
1	10	inf

Conditions III

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
30	98	0:01
	72	0:55
	1	72	5:00
1	10	inf

Tyc B1 2013-08-21

Reaction

what	µl
Bacillus	1
AT03	2
AT04	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	5

Condition

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	56	0:05
	72	0:55
1	72	5:00
1	10	inf

Tyc C5 2013-08-21

what	µl
Bacillus	1
AT05	2
AT06	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	5

Condition

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	61	0:05
	72	0:55
1	72	5:00
1	10	inf

Tyc C6 2013-08-21

what	µl
Bacillus	1
AT07	2
AT08	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	5

Condition

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	56	0:05
	72	0:55
1	72	5:00
1	10	inf

Tyc B1 2013-08-22

Reaction

what	µl
Bacillus	1
AT03	2
AT04	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	5

Conditions II

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
14	98	0:01
	63 ↓0.5	0:05
	72	0:55
16	98	0:01
	58	0:05
	72	0:55
1	72	5:00
1	10	inf

Tyc C5 2013-08-22

Reaction

what	µl
Bacillus	1
AT05	2
AT06	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	4

Conditions II

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
14	98	0:01
	61 ↓0.5	0:05
	72	0:55
16	98	0:01
	63	0:05
	72	0:55
1	72	5:00
1	10	inf

Tyc B1 2013-08-23

B 23-08-2013

Reaction

what	µl
Bacillus	1
AT03	2
AT04	2
Phusion Flash 2x Master Mix	10
DMSO	1.5
ddH20	3.5

Conditions

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	63	0:05
	72	0:50
1	72	5:00
1	10	inf

Tyc C5 2013-08-23

B 23-08-2013

Reaction I

what	µl
Bacillus	1
AT05	2
AT06	2
Phusion Flash 2x Master Mix	10
DMSO	1.5
ddH20	3.5

Reaction II

what	µl
Bacillus	1
AT05	2
AT06	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	4

Conditions

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	63	0:05
	72	0:50
1	72	5:00
1	10	inf