Team:HokkaidoU Japan/Notebook/September2
From 2010.igem.org
(Difference between revisions)
(→Ligation) |
(→RFPとpSB1C3,RFPとpUC119のDigestion/ Ligation/ Transformation) |
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- | = | + | =Digestion, Ligation and Transformation of pUC119, RFP, pSB1C3 and RFP= |
+ | |||
{|style="text-align:center; class="protocol" | {|style="text-align:center; class="protocol" | ||
|- | |- | ||
Line 19: | Line 20: | ||
|35 uL | |35 uL | ||
|- | |- | ||
- | | | + | |pUC119(pre cut) |
|10 ng/uL | |10 ng/uL | ||
|3000 bp | |3000 bp | ||
Line 26: | Line 27: | ||
==Digestion== | ==Digestion== | ||
+ | |||
{|style="text-align:center; float: left;" class="protocol" | {|style="text-align:center; float: left;" class="protocol" | ||
!Reagent | !Reagent | ||
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|} | |} | ||
<div style="clear:both;"></div> | <div style="clear:both;"></div> | ||
- | + | →Incubated at 37C for 60 min<br> | |
- | + | →Adde 10 uL 6x Sample buffer and electrophoresed | |
- | == | + | ==Electrophoresis and gel extraction== |
- | + | To check concentration lanes 2 through 5 contained 1uL of DNA solution | |
- | [[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|]] | + | [[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|Electrophoresis of digestion product]] |
{|class="protocol" | {|class="protocol" | ||
|'''Lane''' | |'''Lane''' | ||
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|- | |- | ||
|1 | |1 | ||
- | | | + | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''d III, EcoR I] |
|- | |- | ||
|2 | |2 | ||
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|blank | |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== | ==Ligation== | ||
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|} | |} | ||
<div style="clear:both;"></div> | <div style="clear:both;"></div> | ||
- | + | →Incubated 16C for min<br> | |
- | + | →Transformed | |
=HSP, RBS, RFP, dTのゲル抽= | =HSP, RBS, RFP, dTのゲル抽= |
Revision as of 17:21, 27 September 2010
Digestion, Ligation and Transformation of pUC119, RFP, pSB1C3 and RFP
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 | Lambda/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
→Transformed