Team:HokkaidoU Japan/Notebook/September14
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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September13|September 13]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div></div> | {{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September13|September 13]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div></div> | ||
- | =pSB1C3, araC Promoter | + | =Digestion and ligation of pSB1C3, araC Promoter and GFP= |
+ | |||
==Digestion== | ==Digestion== | ||
- | [[Image:HokkaidoU Japan 20100914a.jpg|200px|right|thumb|]] | + | |
- | + | [[Image:HokkaidoU Japan 20100914a.jpg|200px|right|thumb|Electrophoresed digestion product]] | |
- | {|style="text-align:center; float:left | + | |
+ | Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times. | ||
+ | |||
+ | {|style="text-align:center; float:left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
|- | |- | ||
|10x M buffer | |10x M buffer | ||
Line 25: | Line 31: | ||
|3 | |3 | ||
|- | |- | ||
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''Total''' |
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''50 uL''' |
|} | |} | ||
- | {|style="text-align:center; float:left | + | {|style="text-align:center; float:left;" class="protocol" |
+ | !Reagent | ||
+ | !Amount | ||
|- | |- | ||
|10x M buffer | |10x M buffer | ||
Line 48: | Line 56: | ||
|2 | |2 | ||
|- | |- | ||
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''Total''' |
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''50 uL''' |
|} | |} | ||
- | {|style="text-align:center; float:left | + | {|style="text-align:center; float:left;" class="protocol" |
+ | !Reagent | ||
+ | !Amount | ||
|- | |- | ||
|10x M buffer | |10x M buffer | ||
Line 71: | Line 81: | ||
|1 | |1 | ||
|- | |- | ||
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''Total''' |
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''50 uL''' |
|} | |} | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
- | + | →Incubated at 37C for 120 min<br> | |
- | + | →Added Sample Buffer 10 uL and electrophoresed | |
- | * | + | * pSB1C3 band wasn't visible |
- | + | → Other 2 were gel extracted | |
- | * 40 | + | * Diluted that final volume would be 40 uL |
+ | ==[[Team:HokkaidoU_Japan/Protocols|Ethanol precipitation]]== | ||
+ | [[Image:HokkaidoU Japan 20100914b.jpg|200px|right|thumb|Electrophoresis after ethanol precipitation]] | ||
- | + | * Added ethanol acetate[3 M] 4 uL to 40 uL of previously purified DNA solution | |
- | [[ | + | * Added EtOH[100%] 44 uL froze in dry ice |
- | * | + | * Melted and centrifuged at 15000 rpm, 4C 5 min |
- | * | + | * Discarded supernatant and added EtOH[70%] 100 uL rinsed |
- | * | + | * Centrifuged at 15,996rpm, 4C 5 min |
- | * | + | * Discarded supernatant and dried |
- | * | + | * Diluted in 2 uL of TE each |
- | promoter: 125 ng/ | + | *Anticipated concentrations<br> |
- | + | promoter: 125 ng/2uL<br> | |
- | + | GFP: 106 ng/2uL | |
- | + | ||
+ | * pSB1C3 was cut(EcoR1, Pst1) on September 6th | ||
+ | * Did ethanol precipitation and electrophoresed to check concentration | ||
+ | * It was about 20 ng/uL | ||
+ | →Used 2 uL of it | ||
==Ligation== | ==Ligation== | ||
- | {|style="text-align:center;" | + | |
+ | {|style="text-align:center;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
|- | |- | ||
|pSB1C3 | |pSB1C3 | ||
Line 115: | Line 133: | ||
|0.4 uL | |0.4 uL | ||
|- | |- | ||
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''Total''' |
- | |style="border-top:1px solid # | + | |style="border-top:1px solid #996;"|'''12.4 uL''' |
|} | |} | ||
- | + | *50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical) |
Latest revision as of 08:20, 27 October 2010
Digestion and ligation of pSB1C3, araC Promoter and GFP
Digestion
Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.
Reagent | Amount |
---|---|
10x M buffer | 5 uL |
DW | 34 |
pSB1C3 | 1 |
BSA | 5 |
EcoR I | 2 |
Pst I | 3 |
Total | 50 uL |
Reagent | Amount |
---|---|
10x M buffer | 5 uL |
DW | 27 |
Promoter | 8 |
BSA | 5 |
EcoR I | 3 |
Spe I | 2 |
Total | 50 uL |
Reagent | Amount |
---|---|
10x M buffer | 5 uL |
DW | 32 |
GFP | 3 |
BSA | 5 |
Xba I | 4 |
Pst I | 1 |
Total | 50 uL |
→Incubated at 37C for 120 min
→Added Sample Buffer 10 uL and electrophoresed
- pSB1C3 band wasn't visible
→ Other 2 were gel extracted
- Diluted that final volume would be 40 uL
Ethanol precipitation
- Added ethanol acetate[3 M] 4 uL to 40 uL of previously purified DNA solution
- Added EtOH[100%] 44 uL froze in dry ice
- Melted and centrifuged at 15000 rpm, 4C 5 min
- Discarded supernatant and added EtOH[70%] 100 uL rinsed
- Centrifuged at 15,996rpm, 4C 5 min
- Discarded supernatant and dried
- Diluted in 2 uL of TE each
- Anticipated concentrations
promoter: 125 ng/2uL
GFP: 106 ng/2uL
- pSB1C3 was cut(EcoR1, Pst1) on September 6th
- Did ethanol precipitation and electrophoresed to check concentration
- It was about 20 ng/uL
→Used 2 uL of it
Ligation
Reagent | Amount |
---|---|
pSB1C3 | 2 uL |
promoter | 2 uL |
GFP | 2 uL |
Mighty Mix | 6 uL |
T4 ligase | 0.4 uL |
Total | 12.4 uL |
- 50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)