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Team:HokkaidoU Japan/Notebook/August10
From 2010.igem.org
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* Template DNA length is calculated by adding prefix and suffix length | * Template DNA length is calculated by adding prefix and suffix length | ||
** Biobrick Length + Prefix length + Suffix Length | ** Biobrick Length + Prefix length + Suffix Length | ||
| - | * | + | * Because transformation of 3-1E failed, PCR progrma was changed to allow more time for |
==PCR反応液の調整 == | ==PCR反応液の調整 == | ||
Revision as of 05:35, 20 September 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
シングルコロニーアイソレーション
コロニーを確認したところ，3-1Eにはコロニーが見られなかった
ほかのプレートもコロニー数が少なかったため，手順に不完全なところがあったのかもしれない
- 実験中に沈殿が見られたため
- コンピテントセルの溶解が不十分だった？
- 混ぜ方が不十分だった？
- ２回目の実験で，若干急いだ感じだったので，吸着などが不十分だった？
3-1E以外を新しい培地に移した
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 transformation of 3-1E failed, PCR progrma was changed to allow more time for
PCR反応液の調整
== 今回は4パーツ増幅のためTotal 245 uL作成した．
| 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 |
- 反応液49 uLを４本のPCRチューブに分注し，Templateを1 uLずつ加えた
- それぞれのPCRチューブとTemplate，lengthは
| 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 |
- DNA lengthはパーツ長＋プライマー(49 bp)
- 3-1EはTransformationがうまくいかなかったため
PCR program
| Predenature | 94℃ 2 min |
| Denature | 98℃ 10 sec |
| Extension | 68℃ 30 sec |
| Hold | 4℃ |
- 30 cycle
- 30 sec/kbpなので１cycle30秒で行った
電気泳動
- 増幅したDNAを5 uLとって6xサンプルバッファー1 uLを加えた
- マーカーはpUC119/Hinf1を使用
- エチブロは20 uL使用
