Team:HokkaidoU Japan/Notebook/September2
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- | {{Template:HokkaidoU_Japan}} | + | {{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September1|September 1]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September3|September 3]]</div></div> |
+ | |||
+ | =[[Team:HokkaidoU_Japan/Protocols|Transformation]] of pUC119, RFP, pSB1C3 and RFP= | ||
+ | |||
+ | ==Parts information== | ||
+ | {|style="text-align:center; class="protocol" | ||
+ | |- | ||
+ | !DNA | ||
+ | !Concentration | ||
+ | !Length | ||
+ | !Amount | ||
+ | |- | ||
+ | |RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) | ||
+ | |80 ng/uL | ||
+ | |1000 bp | ||
+ | |26 uL | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |20 ng/uL | ||
+ | |2000 bp | ||
+ | |35 uL | ||
+ | |- | ||
+ | |pUC119(pre cut) | ||
+ | |10 ng/uL | ||
+ | |3000 bp | ||
+ | |28 uL | ||
+ | |} | ||
+ | |||
+ | ==Digestion== | ||
+ | |||
+ | {|style="text-align:center; float: left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) | ||
+ | |26 uL | ||
+ | |- | ||
+ | |DW | ||
+ | |9 uL | ||
+ | |- | ||
+ | |0.1% BSA | ||
+ | |5 uL | ||
+ | |- | ||
+ | |10x M Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |EcoR I | ||
+ | |2 uL | ||
+ | |- | ||
+ | |Pst I | ||
+ | |3 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''50 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; float: left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |33 uL | ||
+ | |- | ||
+ | |DW | ||
+ | |2 uL | ||
+ | |- | ||
+ | |0.1% BSA | ||
+ | |5 uL | ||
+ | |- | ||
+ | |10x M Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |EcoR I | ||
+ | |2 uL | ||
+ | |- | ||
+ | |Pst I | ||
+ | |3 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''50 uL''' | ||
+ | |} | ||
+ | <div style="clear:both;"></div> | ||
+ | →Incubated at 37C for 60 min<br> | ||
+ | →Adde 10 uL 6x Sample buffer and electrophoresed | ||
+ | |||
+ | ==Electrophoresis and [[Team:HokkaidoU_Japan/Protocols|gel extraction]]== | ||
+ | To check concentration lanes 2 through 5 contained 1uL of DNA solution | ||
+ | [[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|Electrophoresis of digestion product]] | ||
+ | {|class="protocol" | ||
+ | |'''Lane''' | ||
+ | |'''DNA''' | ||
+ | |- | ||
+ | |1 | ||
+ | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III, EcoR I] | ||
+ | |- | ||
+ | |2 | ||
+ | |50 base ladder | ||
+ | |- | ||
+ | |3 | ||
+ | |Heat shock promotor | ||
+ | |- | ||
+ | |4 | ||
+ | |RBS | ||
+ | |- | ||
+ | |5 | ||
+ | |RFP protein coding | ||
+ | |- | ||
+ | |6 | ||
+ | |double Terminator | ||
+ | |- | ||
+ | |7 | ||
+ | |blank | ||
+ | |- | ||
+ | |8 | ||
+ | |style="border-left:1px solid #000;" rowspan="4"|RFP reporter ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) 15 uL each | ||
+ | |- | ||
+ | |9 | ||
+ | |- | ||
+ | |10 | ||
+ | |- | ||
+ | |11 | ||
+ | |- | ||
+ | |12 | ||
+ | |blank | ||
+ | |- | ||
+ | |13 | ||
+ | |rowspan="4" style="border-left:1px solid #000;"|pSB1C3 15 uL each | ||
+ | |- | ||
+ | |14 | ||
+ | |- | ||
+ | |15 | ||
+ | |- | ||
+ | |16 | ||
+ | |- | ||
+ | |17 | ||
+ | |blank | ||
+ | |} | ||
+ | Bands were exised (210 mg and 220 mg acordingly) | ||
+ | Final volume was made to be 20 uL, with disregard to promega's protocol<br> | ||
+ | At this point concentrations were: RFP 104 ng/uL,pSB1C3 33 ng/uL. | ||
+ | |||
+ | ==Ligation== | ||
+ | {|style="text-align:center;float: left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |RFP | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |ligation Kit I | ||
+ | |3 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''6.5 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; float: left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |RFP | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |Mighty Mix | ||
+ | |3 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''6.5 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; float: left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |RFP | ||
+ | |0.8 uL | ||
+ | |- | ||
+ | |pUC119 | ||
+ | |4 uL | ||
+ | |- | ||
+ | |ligation Kit I | ||
+ | |4.8 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''10.5 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; float: left;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |RFP | ||
+ | |0.8 uL | ||
+ | |- | ||
+ | |pUC119 | ||
+ | |4 uL | ||
+ | |- | ||
+ | |Mighty Mix | ||
+ | |4.8 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''10.5 uL''' | ||
+ | |} | ||
+ | <div style="clear:both;"></div> | ||
+ | →Incubated 16C for min<br> | ||
+ | →[[Team:HokkaidoU_Japan/Protocols|Transformation]] |
Latest revision as of 08:02, 27 October 2010
Transformation of pUC119, RFP, pSB1C3 and RFP
Parts information
DNA | Concentration | Length | Amount |
---|---|---|---|
RFP (1-5A) | 80 ng/uL | 1000 bp | 26 uL |
pSB1C3 | 20 ng/uL | 2000 bp | 35 uL |
pUC119(pre cut) | 10 ng/uL | 3000 bp | 28 uL |
Digestion
Reagent | Amount |
---|---|
RFP (1-5A) | 26 uL |
DW | 9 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2 uL |
Pst I | 3 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
pSB1C3 | 33 uL |
DW | 2 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2 uL |
Pst I | 3 uL |
Total | 50 uL |
→Incubated at 37C for 60 min
→Adde 10 uL 6x Sample buffer and electrophoresed
Electrophoresis and gel extraction
To check concentration lanes 2 through 5 contained 1uL of DNA solution
Lane | DNA |
1 | λ/Hind III, EcoR I |
2 | 50 base ladder |
3 | Heat shock promotor |
4 | RBS |
5 | RFP protein coding |
6 | double Terminator |
7 | blank |
8 | RFP reporter (1-5A) 15 uL each |
9 | |
10 | |
11 | |
12 | blank |
13 | pSB1C3 15 uL each |
14 | |
15 | |
16 | |
17 | blank |
Bands were exised (210 mg and 220 mg acordingly)
Final volume was made to be 20 uL, with disregard to promega's protocol
At this point concentrations were: RFP 104 ng/uL,pSB1C3 33 ng/uL.
Ligation
Reagent | Amount |
---|---|
RFP | 1.5 uL |
pSB1C3 | 1.5 uL |
ligation Kit I | 3 uL |
T4 ligase | 0.5 uL |
Total | 6.5 uL |
Reagent | Amount |
---|---|
RFP | 1.5 uL |
pSB1C3 | 1.5 uL |
Mighty Mix | 3 uL |
T4 ligase | 0.5 uL |
Total | 6.5 uL |
Reagent | Amount |
---|---|
RFP | 0.8 uL |
pUC119 | 4 uL |
ligation Kit I | 4.8 uL |
T4 ligase | 0.5 uL |
Total | 10.5 uL |
Reagent | Amount |
---|---|
RFP | 0.8 uL |
pUC119 | 4 uL |
Mighty Mix | 4.8 uL |
T4 ligase | 0.5 uL |
Total | 10.5 uL |
→Incubated 16C for min