Team:HokkaidoU Japan/Notebook/September2
From 2010.igem.org
(Difference between revisions)
(2 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
{{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> | {{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" | {|style="text-align:center; class="protocol" | ||
|- | |- | ||
Line 10: | Line 11: | ||
!Amount | !Amount | ||
|- | |- | ||
- | |RFP ([[Team:HokkaidoU_Japan/ | + | |RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) |
|80 ng/uL | |80 ng/uL | ||
|1000 bp | |1000 bp | ||
Line 32: | Line 33: | ||
!Amount | !Amount | ||
|- | |- | ||
- | |RFP ([[Team:HokkaidoU_Japan/ | + | |RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) |
|26 uL | |26 uL | ||
|- | |- | ||
Line 83: | Line 84: | ||
→Adde 10 uL 6x Sample buffer and electrophoresed | →Adde 10 uL 6x Sample buffer and electrophoresed | ||
- | ==Electrophoresis and gel extraction== | + | ==Electrophoresis and [[Team:HokkaidoU_Japan/Protocols|gel extraction]]== |
To check concentration lanes 2 through 5 contained 1uL of DNA solution | To check concentration lanes 2 through 5 contained 1uL of DNA solution | ||
[[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|Electrophoresis of digestion product]] | [[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|Electrophoresis of digestion product]] | ||
Line 91: | Line 92: | ||
|- | |- | ||
|1 | |1 | ||
- | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png | + | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III, EcoR I] |
|- | |- | ||
|2 | |2 | ||
Line 112: | Line 113: | ||
|- | |- | ||
|8 | |8 | ||
- | |style="border-left:1px solid #000;" rowspan="4"|RFP reporter ([[Team:HokkaidoU_Japan/ | + | |style="border-left:1px solid #000;" rowspan="4"|RFP reporter ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) 15 uL each |
|- | |- | ||
|9 | |9 | ||
Line 136: | Line 137: | ||
|} | |} | ||
Bands were exised (210 mg and 220 mg acordingly) | Bands were exised (210 mg and 220 mg acordingly) | ||
- | Final volume was made to be 20 uL, with disregard to promega | + | 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. | At this point concentrations were: RFP 104 ng/uL,pSB1C3 33 ng/uL. | ||
Line 221: | Line 222: | ||
<div style="clear:both;"></div> | <div style="clear:both;"></div> | ||
→Incubated 16C for min<br> | →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