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Team:HokkaidoU Japan/Notebook/August11
From 2010.igem.org
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 System
From PCR products we only used No.3 （1-23L（terminator）178 bp made on previous day
| Tube No. | 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 |
| Restriction Enzyme Digestion | 4C | at 37C for 60 min | ||
| Restriction enzyme Inactivation | at 60C for 15 min | |||
| ligation solution | 10 uL | 10 uL | 10 uL | 10 uL |
| Ligation | at 16C for 30 min | |||
| 6x SB | 4 uL | 4 uL | 4 uL | 4 uL |
→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOH
Electrophoresis
- Performed electrophoresis for every digested sample, 1 through 4
- Used marker pUC119/Hinf
- DNA solution was too diluted and no bands were visible
