"
Team:HokkaidoU Japan/Notebook/August27
From 2010.igem.org
| Line 3: | Line 3: | ||
=ラオリ英語添削お願い= | =ラオリ英語添削お願い= | ||
| - | =Transfer [[Team:HokkaidoU_Japan/Materials_And_Methods# | + | =Transfer [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] on pSB1A10 to pSB1C3= |
1.estimated concentrations of pSB1C3 and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] which was amplified by miniprep before | 1.estimated concentrations of pSB1C3 and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] which was amplified by miniprep before | ||
Revision as of 11:30, 16 October 2010
since 13 Jul 2010
since 1 Aug 2010
ラオリ英語添削お願い
Transfer 1-1A on pSB1A10 to pSB1C3
1.estimated concentrations of pSB1C3 and 1-1A which was amplified by miniprep before
2.mixed solutions according to the table below
| Reagent | Amount |
|---|---|
| pSB1C3 | 5 uL |
| DW | 75 uL |
| 10x M Buffer | 10 uL |
| 1%BSA | 10 uL |
| EcoRI | 5 uL |
| PstI | 5 uL |
| Total | 100 uL |
| Reagent | Amount |
|---|---|
| 1-1A | 8 uL |
| DW | 6 uL |
| 10x M Buffer | 2 uL |
| 1%BSA | 2 uL |
| EcoRI | 1 uL |
| PstI | 1 uL |
| Total | 20 uL |
3.incubated at 37C for 60min
4.purified the two samples
5.added 250 uL 100% EtOH to each samples
6.centrifuged at 4C, 15000rpm for 10 min
7.discarded supernatant
8.put the samples into desiccator
9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A
10.mixed 1 uL pSB1C3 and 2 uL 1-1A
11.added 3 uL ligation solution and 0.5 uL T4 ligase
12.transfered the sample to a tube of 500 uL
13.incubated at 16C for 30 min
->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.
Preparation of 3 Piece Ligation
- Parts Information
| Name | Biobrick Name | Well | Length |
|---|---|---|---|
| Heat Sensor | BBa_K098995 | 3-1E | 937 bp |
| RBS | BBa_B0034 | 1-2M | 12 bp |
| RFP | BBa_E1010 | 1-18F | 681 bp |
| Double Terminator | BBa_0015 | 1-23L | 129 bp |
1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator
2.mixed solutions according to the table below
| Reagent | Amount |
|---|---|
| DW | 13.5 uL |
| 10x M buffer | 5 uL |
| 1%BSA | 5 uL |
| EcoRI | 3 uL |
| SpeI | 1 uL |
| Heat Sensor | 22.5 uL |
| Total | 50 uL |
| Reagent | Amount |
|---|---|
| DW | 34.9 uL |
| 10x M buffer | 5 uL |
| 1%BSA | 5 uL |
| XbaI | 1 uL |
| PstI | 2 uL |
| RBS | 34.9 uL |
| Total | 50 uL |
| Reagent | Amount |
|---|---|
| DW | 25.5 uL |
| 10x M buffer | 5 uL |
| 1%BSA | 5 uL |
| EcoRI | 3 uL |
| SpeI | 1 uL |
| RFP | 10.5 uL |
| Total | 50 uL |
| Reagent | Amount |
|---|---|
| DW | 34 uL |
| 10x M buffer | 5 uL |
| 1%BSA | 5 uL |
| XbaI | 1 uL |
| PstI | 2 uL |
| Double Terminator | 3 uL |
| Total | 50 uL |
