Team:HokkaidoU Japan/Notebook/September16

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*digestion of GFP(1-14K) and double terminator(1-23L)
 
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== Digestion of GFP and Double Terminator ==
 
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Parts Information
 
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GFP : BBa_E0040 1-14K 720bp pSB1A3
 
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double terminator : BBa_B0015 1-23L 129bp pSB1AK3
 
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pSB1A3
 
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1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
 
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Method
 
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1)electrophoresed 1μl of 1-14K and 1-23L added 0.5μl of 6×sample buffer to estimate concentration of each solution.
 
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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.
 
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estimates of concentration
 
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1-14K:200ng/μl
 
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1-23L:120ng/μl
 
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pSB1A3:2.5ng/μl
 
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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.
 
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1-23L0.5μl
 
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M-buffer5 μl
 
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0.1%BSA5　μl
 
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Xba4 μl 
 
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Pst0.5μl 
 
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DW35μl
 
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1-14K1.5μl
 
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H-buffer2 μl
 
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0.1%BSA2 μl
 
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Eco1 μl
 
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Spe0.5μl
 
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DW13μl
 
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pSB1A320μl
 
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H-buffer3μl
 
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0.1%BSA3 μl
 
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Eco0.5 μl
 
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Pst0.5μl
 
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DW3μl
 
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pSB1A330μl
 
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H-buffer5μl
 
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0.1%BSA5μl
 
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Eco0.5 μl
 
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Pst0.5μl
 
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DW9μl
 
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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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5)electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.
 
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1:λ/HindⅢ　EcoRⅠ
 
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2~3:1-14K
 
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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℃.
 

Latest revision as of 17:35, 19 September 2010

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