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Team:Tokyo Metropolitan/Notebook/Fiber/2010/10/23
From 2010.igem.org
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*ethanol | *ethanol | ||
*EDTA | *EDTA | ||
| + | *Hi-Di solution | ||
'''procedure''' | '''procedure''' | ||
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#PCR | #PCR | ||
#Add EDTA and Ethanol and put at room temperature (15min) | #Add EDTA and Ethanol and put at room temperature (15min) | ||
| - | #centrifuge (8000rpm,30min) | + | #centrifuge (8000rpm,30min) and throw away supernatant |
| - | + | ||
#add ethanol again | #add ethanol again | ||
| - | #centrifuge (8000rpm,15min) | + | #centrifuge (8000rpm,15min) and throw away supernatant |
| - | + | ||
| - | + | ||
#add Hi-Di solution | #add Hi-Di solution | ||
| + | #heat at 95℃ | ||
#transfer these sample to plate for sequence | #transfer these sample to plate for sequence | ||
#read sequence | #read sequence | ||
| + | '''result''' | ||
| + | bcsA wasn't inserted into pSB1C3... | ||
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===Experiment:Colony PCR=== | ===Experiment:Colony PCR=== | ||
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seem to be correct inserts | seem to be correct inserts | ||
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<br/> | <br/> | ||
Revision as of 16:51, 25 October 2010
E.coli Fiber Project Notebook
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2010/10/23 Saturday (Naoto)
member
naoto and watachin
Experiment:Sequence
material
For PCR
- bcsA (No.9)
- Big Dye
- primer
- DW
For Ethanol precipitation
- ethanol
- EDTA
- Hi-Di solution
procedure
- mix materials(DNA<50ng)
- PCR
- Add EDTA and Ethanol and put at room temperature (15min)
- centrifuge (8000rpm,30min) and throw away supernatant
- add ethanol again
- centrifuge (8000rpm,15min) and throw away supernatant
- add Hi-Di solution
- heat at 95℃
- transfer these sample to plate for sequence
- read sequence
result bcsA wasn't inserted into pSB1C3...
Experiment:Colony PCR
material
- colony of E.coli
- bcsB:1~32
- bcsC:33~64
- bcsD:65~96
other materials were same as protocol3
procedure
see protocol3
result
From the length of bands
bcsC→No.54 and 64
bcsD→No.75
seem to be correct inserts
