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Team:HokkaidoU Japan/Notebook/August23
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=pSB1C3 Activity Check= | =pSB1C3 Activity Check= | ||
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*Used 2 uL of from 100 uL of gel extracted DNA for liagation | *Used 2 uL of from 100 uL of gel extracted DNA for liagation | ||
** Namely added 2 uL of Ligation Solution to 2 uL of DNA | ** Namely added 2 uL of Ligation Solution to 2 uL of DNA | ||
| - | *Incubated at 16C for | + | *Incubated at 16C for 30 min |
===Ligation Negative Control=== | ===Ligation Negative Control=== | ||
Latest revision as of 08:30, 27 October 2010
since 13 Jul 2010
since 1 Aug 2010
pSB1C3 Activity Check
EcoR I/Pst I Digestion
| Reagent | Amount |
| pSB1C3 | 50 uL |
| DW | 4 uL |
| 0.1% BSA | 7 uL |
| 10x M Buffer | 7 uL |
| EcoR I | 1 uL |
| Pst I | 1 uL |
| Total | 70 uL |
- Incubated at 37C for 60 min
- Added 15 uL of 6x Sample Buffer and electrophoresed on six lanes
- Extracted from gel
Ligated
- Used 2 uL of from 100 uL of gel extracted DNA for liagation
- Namely added 2 uL of Ligation Solution to 2 uL of DNA
- Incubated at 16C for 30 min
Ligation Negative Control
Used 2 uL of from 100 uL of gel extracted DNA for Transformation
Vector PCR: We meet again!
Amplified pSB1A3, pSB1C3 and pSB1T3 by PCR as listed in the table bellow.
| Reagent | Amount |
| Vector | 1 |
| DW | 33 |
| 10x Buffer | 5 |
| 2 M 4dNTPs | 5 |
| 25 mM MgSO4 | 3 |
| Suffix-F | 1 |
| Prefix-R | 1 |
| KOD | 1 |
| Total | 50 uL |
- Only deviation from protocol was increase to extention to 120 sec
