Team:HokkaidoU Japan/Notebook/September16

• digestion of GFP(1-14K) and double terminator(1-23L)

Digestion of GFP and Double Terminator

Parts Information

GFP	BBa_E0040	1-14K	720bp	pSB1A3
double terminator	BBa_B0015	1-23L	129bp	pSB1AK3
pSB1A3	pSB1A3	-	2157bp	pSB1A3

1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.

Method

1. electrophoresed 1μl of 1-14K and 1-23L added 0.5μl of 6×sample buffer to estimate concentration of each solution.
2. estimated concentration from photo of electrophoresys.But I forgot to electrophorese pSB1A3 solution with the other samples,so pSB1A3 solution was done by other person.
3. made digestion recipes based on each concentrations.Why pSB1A3 recipe is two,because I firstly made 30μl of pSB1A3 solution,but I found it was insufficient to ligate parts,so I made more 50μl of it after.

Estimates of concentration

1-14K:200ng/μl

1-23L:120ng/μl

pSB1A3:2.5ng/μl

Digestion Menu

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

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

1.5μl

H-buffer2 μl

0.1%BSA2 μl

Eco1 μl

Spe0.5μl

DW13μl

pSB1A320μl

H-buffer3μl

0.1%BSA3 μl

Eco0.5 μl

Pst0.5μl

DW3μl

pSB1A330μl

H-buffer5μl

0.1%BSA5μl

Eco0.5 μl

Pst0.5μl

DW9μl

4)put each solutions into 37℃ incubator.1-23L solution was put about a half and two hours,1-14K solution was done about a half and an hour,30μl of pSB1A3 solution was done about an hour,and 50μl of pSB1A3 was done about a half hour.

5)electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.

1:λ/HindⅢ　EcoRⅠ

2~3:1-14K

4~8:1-23L

9~16:pSB1A3

6)cannot see 1-23L because of overflowing.extracted the other samples from a gel.dissolve them with 50μl of Nuclease free water.And they were stocked to freeze in -20℃.