Team:HokkaidoU Japan/Notebook/September17

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Construction of GFP marker for a part which will be secreted using T3SS
Ordered primers for construction for same part

Digestion of GFP and Double Terminator

Parts Information

Description	BioBrick No.	Well No.	Length	Plasmid
GFP	BBa_E0040	1-14K	720bp	pSB1A3
double terminator	BBa_B0015	1-23L	129bp	pSB1AK3
pSB1A3	pSB1A3		2157bp	pSB1A3

Parts in wells 1-14K and pSB1A3 were purified with mycrocon. Part 1-23L was extracted from a gel previously.

Performed electrophoresis of 1-14K and 1-23L to estimate concentration of each solution.
Estimated concentration from photo of electrophoresis
pSB1A3 solution was done by other person.
Made digestion recipes(below) based on estimated concentrations
Made 30 ul of pSB1A3 solution, but latter found it insufficient to ligate parts
- made more 50ul of it after

Part Well No.	Amount
1-14K	200 ng/ul
1-23L	120 ng/ul
pSB1A3	2.5 ng/ul

Digestion

Reagent	Amount
1-23L	0.5 uL
10x M buffer	5 uL
0.1%BSA	5 uL
Xba I	4 uL
Pst I	0.5 uL
DW	35 uL
Total	50 uL

Reagent	Amount
1-14K	1.5 uL
10x H buffer	2 uL
0.1% BSA	2 uL
EcoR I	1 uL
Spe I	0.5 uL
DW	13 uL
Total	20 uL

Reagent	Amount
pSB1A3	20 uL
10x H buffer	3 uL
0.1% BSA	3 uL
EcoR I	0.5 uL
Pst I	0.5 uL
DW	3 uL
Total	30 uL

Reagent	Amount
pSB1A3	30 uL
10x H buffer	5 uL
0.1% BSA	5 uL
EcoR I	0.5 uL
Pst I	0.5 uL
DW	9 uL
Total	30 uL

Incubated each solution at 37C
Solution of 1-23L was incubated for 150 min
Solution of 1-14K was incubated for 90 min
30 ul of pSB1A3 solution was incubated for 60 min
50 ul of pSB1A3 solution was incubated for 30 min
Performed electrophoresis for each solution

put 12 uls each into wells of a gel like below.

Lane	DNA
1	λ/HindIII,　EcoR I
2~3	1-14K
4~8	1-23L
9~16	pSB1A3

Results

Could not see bands of 1-23L because leaked out
- Drove current for too long
Extracted the other samples from a gel using promega kit
Stored all at -20C.

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