Team:HokkaidoU Japan/Notebook/September17

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== Digestion of GFP and Double Terminator ==
 +
===Parts Information===
 +
{| class="protocol"
 +
|-
 +
|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.
 +
 
 +
 
 +
* electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer to estimate concentration of each solution.
 +
* 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.
 +
* made digestion recipes based on each concentrations(below). Why pSB1A3 recipe is two, because I firstly made 30ul of pSB1A3 solution, but I found it was insufficient to ligate parts, so I made more 50ul of it after.
 +
 
 +
{|style="text-align:center;" class="protocol"
 +
|-
 +
|1-14K
 +
|200 ng/ul
 +
|-
 +
|1-23L
 +
|120 ng/ul
 +
|-
 +
|pSB1A3
 +
|2.5 ng/ul
 +
|}
 +
 
 +
===Digestion Menu===
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
|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
 +
|-
 +
|style="border-top:1px solid #000;"|'''Total'''
 +
|style="border-top:1px solid #000;"|'''50 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
|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
 +
|-
 +
|style="border-top:1px solid #000;"|'''Total'''
 +
|style="border-top:1px solid #000;"|'''20 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
|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
 +
|-
 +
|style="border-top:1px solid #000;"|'''Total'''
 +
|style="border-top:1px solid #000;"|'''30 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
|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
 +
|-
 +
|style="border-top:1px solid #000;"|'''Total'''
 +
|style="border-top:1px solid #000;"|'''30 uL'''
 +
|}
 +
 
 +
<div style="clear:both"></div>
 +
 
 +
* put each solutions into 37C incubator.
 +
* 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour.
 +
* electrophoresed each solutions added 6x sample buffer.
 +
* put 12uls each into wells of a gel like below.
 +
 
 +
{|class="protocol"
 +
|-
 +
|1
 +
|λ/''Hin''dIII,　EcoR I
 +
|-
 +
|2~3
 +
|1-14K
 +
|-
 +
|4~8
 +
|1-23L
 +
|-
 +
|9~16
 +
|pSB1A3
 +
|}
 +
 
 +
'''Result'''
 +
* cannot see 1-23L because of overflowing.
 +
* extracted the other samples from a gel.
 +
* dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.

Revision as of 17:35, 19 September 2010

Notebook

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.


  • electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer to estimate concentration of each solution.
  • 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.
  • made digestion recipes based on each concentrations(below). Why pSB1A3 recipe is two, because I firstly made 30ul of pSB1A3 solution, but I found it was insufficient to ligate parts, so I made more 50ul of it after.
1-14K 200 ng/ul
1-23L 120 ng/ul
pSB1A3 2.5 ng/ul

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
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
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
  • put each solutions into 37C incubator.
  • 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour.
  • electrophoresed each solutions added 6x sample buffer.
  • put 12uls each into wells of a gel like below.
1 λ/HindIII,　EcoR I
2~3 1-14K
4~8 1-23L
9~16 pSB1A3

Result

  • cannot see 1-23L because of overflowing.
  • extracted the other samples from a gel.
  • dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September17"