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Team:HokkaidoU Japan/Notebook/August10
From 2010.igem.org
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==Single Colony Isolation== | ==Single Colony Isolation== | ||
* Observed if any colonies were made | * Observed if any colonies were made | ||
| - | ** Could not see [[Team:HokkaidoU_Japan/ | + | ** Could not see [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]] |
* Number of other colonies on other plates was also very small | * Number of other colonies on other plates was also very small | ||
** Mistake in protocol is inferred | ** Mistake in protocol is inferred | ||
** Precipitation of cells maybe at fault | ** Precipitation of cells maybe at fault | ||
| - | * All but [[Team:HokkaidoU_Japan/ | + | * All but [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]] was moved to new plates |
==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System== | ==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System== | ||
| Line 54: | Line 54: | ||
|- | |- | ||
| 1 | | 1 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|2-24G]]（sender） |
| 847 bp | | 847 bp | ||
|- | |- | ||
| 2 | | 2 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-2M]]（RBS） |
| 61 bp | | 61 bp | ||
|- | |- | ||
| 3 | | 3 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-23L]]（terminator） |
| 178 bp | | 178 bp | ||
|- | |- | ||
| 4 | | 4 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]]（heat sensor） |
| 984 bp | | 984 bp | ||
|} | |} | ||
| Line 108: | Line 108: | ||
|- | |- | ||
| 2 | | 2 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|2-24G]]（sender） |
| 847 bp | | 847 bp | ||
|- | |- | ||
| 3 | | 3 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-2M]]（RBS） |
| 61 bp | | 61 bp | ||
|- | |- | ||
| 4 | | 4 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-23L]]（terminator） |
| 178 bp | | 178 bp | ||
|- | |- | ||
| 5 | | 5 | ||
| - | | [[Team:HokkaidoU_Japan/ | + | | [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]]（heat sensor） |
| 984 bp | | 984 bp | ||
|} | |} | ||
Revision as of 11:24, 16 October 2010
since 13 Jul 2010
since 1 Aug 2010
Single Colony Isolation
- Observed if any colonies were made
- Could not see 3-1E
- Number of other colonies on other plates was also very small
- Mistake in protocol is inferred
- Precipitation of cells maybe at fault
- All but 3-1E was moved to new plates
PCR Reaction System
Reaction system was prepared for 3 parts, 245 uL in total
| Reagent | Amount |
|---|---|
| Autoclaved DW | 165 uL |
| 10x PCR buffer | 25 uL |
| 2 mM dNTPs | 25 uL |
| 25 mM MgSO4 | 15 uL |
| EX-F primer | 5 uL |
| PS-R primer | 5 uL |
| KOD plus Neo | 5 uL |
| Total | 245 uL |
- 49 uL of reaction solution was added to each of 4 tubes and after DNA template was added
- Length of each template is listed int the table below
| Tube | Biobrick | Length |
|---|---|---|
| 1 | 2-24G（sender） | 847 bp |
| 2 | 1-2M（RBS） | 61 bp |
| 3 | 1-23L（terminator） | 178 bp |
| 4 | 3-1E（heat sensor） | 984 bp |
- Template DNA length is calculated by adding prefix and suffix length
- Biobrick Length + Prefix length + Suffix Length
- Because Mini prep of 3-1E failed, it amplified by PCR
PCR program
| Predenature | 94℃ 2 min |
| Denature | 98℃ 10 sec |
| Extension | 68℃ 30 sec |
| Hold | 4℃ |
- 30 cycles
- KOD potency is 30 sec/kbp, so 30 sec for１cycle
Electrophoresis
- Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
- Used marker pUC1d19/Hinf1
- Added 20 uL of EtBr to 1/2 TBE
| Lane | DNA | Length of DNA |
|---|---|---|
| 1 | pUC1d19/Hinf1 | |
| 2 | 2-24G（sender） | 847 bp |
| 3 | 1-2M（RBS） | 61 bp |
| 4 | 1-23L（terminator） | 178 bp |
| 5 | 3-1E（heat sensor） | 984 bp |
