Team:HokkaidoU Japan/Notebook/September2
From 2010.igem.org
(Difference between revisions)
Line 11: | Line 11: | ||
!Amount | !Amount | ||
|- | |- | ||
- | |RFP ([[Team:HokkaidoU_Japan/ | + | |RFP ([[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-5A]]) |
|80 ng/uL | |80 ng/uL | ||
|1000 bp | |1000 bp | ||
Line 33: | Line 33: | ||
!Amount | !Amount | ||
|- | |- | ||
- | |RFP ([[Team:HokkaidoU_Japan/ | + | |RFP ([[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-5A]]) |
|26 uL | |26 uL | ||
|- | |- | ||
Line 113: | 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/Materials_And_Methods#BioBricks|1-5A]]) 15 uL each |
|- | |- | ||
|9 | |9 |
Revision as of 11:31, 16 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 | 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