Team:Chiba/jk/β/week 4

From 2008.igem.org

Week 4

>last week

7 September, 2008

Sample DNA 1 : BBa_R0010(unknown)[1]

Sample DNA 2 : BBa_R0010(100ng/μL)[2]

Sample No	1,2
Sample DNA	12
PstⅠ	0.5
SpeⅠ	0.5
Buffer 2	2
(×10)BSA	2
dH₂O	3
TOTAL	20

--->Gel Check

Sample	left	right
Sample DNA	5	5
Loading Dye	1	1
TOTAL	6	6

Left side:BBa_R0010 Sample No.1

Right side:BBa_R0010 Sample No.2

-->(8/9)

8 September, 2008

-->(7/9)

Gel Extract

--->Zymo Clean

->15μL?

9 September, 2008

-->(6/9)PCR of I9030

--->Gel Check

--->Gel Extract

--->Zymo Clean

10 September, 2008

-->(10/9)

Colony Count

LuxI
LuxI with LVA
Las
Background(R0010)
Background(C0261)

Pick colony

Colony-PCR

Colony PCR of 10 colonies from ligation plate(9/9-(2)) and 3 colonies from ligation plate(9/9-(3)) one from control plate(BBa_F2620[3](2007)).

DNA Template	1
dNTP mix	5
Foward Primer	0.3
Reverse Primer	0.3
taq DNA polymerase	0.5
Thermopol Buffer	3
Nuclease free water	20
TOTAL	30μL

??

95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃

??

-->Gel Check

--->(28/8)Miniprep

elute 50μL

11 September, 2008

12 September, 2008

13 September, 2008

Gel Check

BBa_C0161[4](2007)
BBa_J04500[5](2007)
BBa_R0010[6](2007)

Sample No	1~3
Sample DNA	1
Loading Dye	1
dH2O	4
TOTAL	6

From left;

-->Digestion

BBa_C0161[7](2007)
BBa_J04500[8](2007)
BBa_R0010[9](2007)

Sample No	①	②	③	④
Sample DNA	5	5	1	1
EcoRⅠ	0.5	o.5	-	-
SpeⅠ	0.5	0.5	-	-
Buffer 1	1	1	-	-
Buffer 3	-	-	1	-
BSA	1	1	-	-
dH2O	2	2	7.5	-
TOTAL	10	10	10	1

>next week

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