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-	{{Template:HokkaidoU_Japan}}	+	{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September17|September 17]]</div></div>
-	*digestion of GFP(1-14K) and double terminator(1-23L)	+
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-	== Digestion of GFP and Double Terminator ==	+
-	Parts Information	+
-		+
-	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	+
-		+
-	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℃.	+
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-	6)	+

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Latest revision as of 17:35, 19 September 2010