Team:HokkaidoU Japan/Notebook/September16

From 2010.igem.org

(Difference between revisions)
(→Digestion Menu)
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|style="border-top:1px solid #000;"|'''20 uL'''
|style="border-top:1px solid #000;"|'''20 uL'''
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{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
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{|style="text-align:center; float:left;" class="protocol"
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|-
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|pSB1A3
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|30 uL
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|-
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|10x H buffer
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|5 uL
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|-
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|0.1% BSA
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|5 uL
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|-
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|EcoR I
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|0.5 uL
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|-
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|Pst I
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|0.5 uL
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|-
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|DW
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|9 uL
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|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''30 uL'''
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|}
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pSB1A330μl
 
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H-buffer5μl
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# 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.
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0.1%BSA5μl
 
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Eco0.5 μl
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# electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.
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Pst0.5μl
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{|
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|-
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DW9μl
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|1
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|λ/''Hin''dIII,　EcoR I
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|-
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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.
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|2~3
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|1-14K
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|-
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5)electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.
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|4~8
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|1-23L
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1:λ/HindⅢ　EcoRⅠ
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|-
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|9~16
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2~3:1-14K
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|pSB1A3
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|}
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4~8:1-23L
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9~16:pSB1A3
 
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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℃.
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* 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℃.

Revision as of 07:34, 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
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


  1. 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.


  1. electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.
1 λ/HindIII,　EcoR I
2~3 1-14K
4~8 1-23L
9~16 pSB1A3


  • 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℃.
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