Team:HokkaidoU Japan/Notebook/September16

From 2010.igem.org

(Difference between revisions)
(→Digestion of GFP and Double Terminator)
Line 5: Line 5:
== Digestion of GFP and Double Terminator ==
== Digestion of GFP and Double Terminator ==
-
Parts Information
+
==Parts Information==
-
 
+
{| class="protocol"
-
GFP : BBa_E0040 1-14K 720bp pSB1A3
+
|-
-
 
+
|GFP
-
double terminator : BBa_B0015 1-23L 129bp pSB1AK3
+
|BBa_E0040
-
 
+
|1-14K
-
pSB1A3
+
|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.
1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
-
Method
+
==Method==
-
1)electrophoresed 1μl of 1-14K and 1-23L added 0.5μl of 6×sample buffer to estimate concentration of each solution.
+
# electrophoresed 1μl of 1-14K and 1-23L added 0.5μl 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.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.
-
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.
+
==Estimates of concentration==
-
 
+
-
 
+
-
estimates of concentration
+
1-14K:200ng/μl
1-14K:200ng/μl
Line 32: Line 45:
-
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.
+
===Digestion Menu===
-
 
+
{|style="text-align:cente; float:left;" class="protocol"
-
1-23L0.5μl
+
|-
-
 
+
|1-23L
-
M-buffer5 μl
+
|0.5 uL
-
 
+
|-
-
0.1%BSA5　μl
+
|10x M buffer
-
 
+
|5 uL
-
Xba4 μl 
+
|-
-
 
+
|0.1%BSA
-
Pst0.5μl 
+
|5 uL
-
 
+
|-
-
DW35μl
+
|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'''
 +
|}
-
1-14K1.5μl
+
{|style="text-align:cente; 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:cente; float:left;" class="protocol"
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|style="border-top:1px solid #000;"|
 +
|style="border-top:1px solid #000;"|
 +
|}
 +
{|style="text-align:cente; float:left;" class="protocol"
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|
 +
|
 +
|-
 +
|style="border-top:1px solid #000;"|
 +
|style="border-top:1px solid #000;"|
 +
|}
 +
1.5μl
H-buffer2 μl
H-buffer2 μl

Revision as of 07:25, 19 September 2010

Notebook
  • 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℃.
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September16"