From 2013.igem.org
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
|
"
