From 2013.igem.org
RFC10 Standardization of Tyc Modules
new Primer for B1 and a new fw Primer for TycC5
Amplification Tyc B1 and Tyc C5
29-08-2013
- Reaction I
| what | µl
|
| Bacillus | 1
|
| AT09/11 | 2
|
| AT10/06 | 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
|
| 65 ↓0.5 | 0:05
|
| 72 | 0:55
|
| 16 | 98 | 0:01
|
| 68 | 0:05
|
| 72 | 0:55
|
| 1 | 72 | 5:00
|
| 1 | 10 | inf
|
Results of Gel extraction Tyc B1 and Tyc C5
| Sample | concentration ng/µl | Adsorption 260 | Adsorption 280
|
| H2O | 0.1 | 0.002 | 0.015
|
| B1 | 43.5 | 0.871 | 0.461
|
| C5 | 23.9 | 0.477 | 0.296
|
Restriction Digest
Tyc ADeltaCom, B1, C5, C6 (AT_01 to AT02, AT_09 to AT_10, AT_11 to AT_06, AT_07 to AT_08; xx kb; xx kb ; xx kb; xx kb) with EcoRI-HF and PstI
| what | µl
|
| Tyc ADeltaCom, B1, C5, C6 | 20
|
| EcoRI-HF | 1
|
| PstI-HF | 1
|
| Buffer CutSmart | 5
|
| dd H2O | 24
|
Inkubate 37°C 2h
Restriction Digest of pSB1C3 (xx kb) with EcoRI-HF and PstI
| what | µl
|
| pSB1C3 | 3
|
| EcoRI-HF | 0.5
|
| PstI-HF | 0.5
|
| Buffer CutSmart | 5
|
| dd H2O | 41
|
Inkubate 37°C 2h
Nucleotide Removal and Concentration Measurement of digested pSB1C3, Tyc ADeltaCom, B1, C5, C6
Concentration measurements showed that DNA was lost with Nucleotide Removal.
Reamplification and RFC10_Modules#Restriction_Digestas above
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