Team:HokkaidoU Japan/Notebook/September6
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(Difference between revisions)
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- | =Ligation of pSB1C3 PCRed from [[Team:HokkaidoU_Japan/ | + | =Ligation of pSB1C3 PCRed from [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] and [[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]](RFP reporter)= |
* Purified through gel extraction to remove primers | * Purified through gel extraction to remove primers | ||
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!Amount | !Amount | ||
|- | |- | ||
- | |RFP ([[Team:HokkaidoU_Japan/ | + | |RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) |
|0.75 uL | |0.75 uL | ||
|- | |- | ||
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|5 uL | |5 uL | ||
|- | |- | ||
- | |10x M Buffer | + | |10x '''M''' Buffer |
|5 uL | |5 uL | ||
|- | |- | ||
Line 45: | Line 45: | ||
|5 uL | |5 uL | ||
|- | |- | ||
- | |10x M Buffer | + | |10x '''M''' Buffer |
|5 uL | |5 uL | ||
|- | |- | ||
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!Amount | !Amount | ||
|- | |- | ||
- | |RFP ([[Team:HokkaidoU_Japan/ | + | |RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) |
|0.75 uL | |0.75 uL | ||
|- | |- |
Latest revision as of 08:08, 27 October 2010
Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)
- Purified through gel extraction to remove primers
- Digested as listed in the table below
Reagent | Amount |
---|---|
RFP (1-5A) | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
pSB1C3 | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
RFP (1-5A) | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x H Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
pSB1C3 | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x H Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
- Extracted from filter with 25 uL, 50 uL DNase free water
- At this point solution volumes were 22.56 ng/uL,45.12 ng/uL acordingly
- Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
- Ligated
- Transformed to 50 uL of competent cells