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Team:HokkaidoU Japan/Notebook/August11
From 2010.igem.org
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==Ligation== | ==Ligation== | ||
| - | === | + | ===DNA Preparation for Ligation=== |
| - | * | + | * Used 49 uL of yesterdays PCR product |
| - | * | + | * Removed primers via Microcon YM-10 |
| - | + | ** Added 450 uL of TE to make final volume of 500 uL | |
| - | ** 500 | + | ** Centrifuged at 10000G for more than an hour till all 4 samples volume was less then 45 uL |
| - | ** | + | * Measured the final amount of samples and added TE till all were 45 uL |
| - | * | + | ** Used 500 uL tubes |
| - | ** | + | |
===ligation反応系=== | ===ligation反応系=== | ||
Revision as of 06:18, 20 September 2010
since 13 Jul 2010
since 1 Aug 2010
LB Culture
- For every 2 mL of LB added 2 uL of antibiotics
- Transfered a colony to LB
- One colony didn't grow well so we isolated another one
- Prepared more tubes for mini preps int he future
glycerol Stock
- Added 1 mL of 80% Glycerol to screw cap tube
- Added 1 mL of cell and broth solution to Glycerol after 2h of incubation
- Sored at -80℃
Ligation
DNA Preparation for Ligation
- Used 49 uL of yesterdays PCR product
- Removed primers via Microcon YM-10
- Added 450 uL of TE to make final volume of 500 uL
- Centrifuged at 10000G for more than an hour till all 4 samples volume was less then 45 uL
- Measured the final amount of samples and added TE till all were 45 uL
- Used 500 uL tubes
ligation反応系
PCR productsは前日に作成した３番（1-23L（terminator）178 bp）を１つだけ使用した
| １ | ２ | ３ | ４ | |
| PCR products | 1 uL | 1 uL | 1 uL | 1 uL |
| 10x H Buffer | - | 1 uL | 1 uL | 1 uL |
| DW | 9 uL | 7.5 uL | 7.0 uL | 7.5 uL |
| Xba1 | - | 0.5 uL | 0.5 uL | - |
| Pst1 | - | - | 0.5 uL | 0.5 uL |
| 制限酵素処理 | 4℃ | @37℃ 60 min | ||
| 制限酵素不活化 | @60℃ 15 min | |||
| ligation solution | 10 uL | 10 uL | 10 uL | 10 uL |
| Ligation反応 | @16℃ 30 min | |||
| 6x SB | 4 uL | 4 uL | 4 uL | 4 uL |
→1% Agarose Gel Electrophoresis in 1/2 TBE
電気泳動
- 制限酵素処理したサンプル１～４を電気泳動した
- マーカーはpUC119/Hinf
- DNA solutionが薄すぎたため，バンドが見えなかった
