From 2008.igem.org
>送受信班
>Protocols
Week 2
24 August, 2008
- (22/8)--->Mini prep
- BBa_J04500(2007)
- BBa_J04500(2006)
- BBa_J13002(2007)
- BBa_J13002(2006)
- --->Digestion test
- BBa_J04500(2007)
- BBa_J04500(2006)
- BBa_J13002(2007)
- BBa_J13002(2006)
- --->Gel Check
|
Sample No. |
5~8 |
Sample DNA |
9 |
Loading Dye |
2 |
dH2O |
1 |
TOTAL |
12 |
From the left;
(single digestuion)
- 5, BBa_J04500(2007) -> OK
- 6, BBa_J04500(2006) -> OK
- 7, BBa_J13002(2007) -> 3400bp:Bad, 2200bp:OK
- 8, BBa_J13002(2006) -> OK
|
25 August, 2008
- (24/8)--->Digestion
- Double Digestion:BBa_J04500(2007)
- Double Digesiton:BBa_R0010(2007)
- Single Digestion:BBa_R0010(2007)
- BBa_R0010(2007)
Sample No. |
1 | 2 | 3 | 4 |
Sample DNA |
5 | 5 | 1 | 1 |
EcoRⅠ |
0.5 | 0.5 | - | - |
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 |
- --->Gel Check
|
Sample No. |
1~3 | 4 |
Sample DNA |
10 | 1 |
Loading Dye |
2 | 1 |
dH2O |
- | 4 |
TOTAL |
12 | 6 |
- From right;
- Double Digestion:BBa_J04500(2007) -> OK
- Double Digesiton:BBa_R0010(2007) -> ?(low)
- Single Digestion:BBa_R0010(2007) -> ?(low)
- BBa_R0010(2007) -> OK(low)
|
--->Digestion
(Double Digestion)
- BBa_C0178
- BBa_C0170
- BBa_J04500(2007)
Sample No. |
1 | 2 | 3 |
Sample DNA |
33ng/μl×60μl | 33ng/μl×60μl | 100ng/μl×20μl |
PstⅠ |
2 | 2 | 2 |
XbaⅠ |
2 | 2 | - |
SpeⅠ |
- | - | 2 |
Buffer 2 |
- | - | 5 |
Buffer 3 |
10 | 10 | - |
BSA |
10 | 10 | 5 |
dH2O |
16 | 16 | 16 |
TOTAL |
100 | 100 | 52 |
26 August, 2008
- (8/25)--->Gel Check
|
Sample No. |
1~3 |
Sample DNA |
30 |
Loading Dye |
6 |
TOTAL |
36 |
- 1, BBa_C0178
- Insert:609bp -> OK
|
--->Gel Extract
- --->Zymo Clean
- 5μl
- 5μl
- 27μl
- --->Gel Check
|
Sample No. |
1~3 |
Sample DNA |
2 |
Loading Dye |
2 |
H2O |
8 |
TOTAL |
12 |
- From left;
- insert:BBa_C0178 --> OK
- insert:BBa_C0170 --> OK?
- vector:BBa_J04500 --> OK
|
- --->SAP
Sample No. |
3 |
Sample DNA |
26 |
SAP |
1 |
SAP Buffer |
3 |
TOTAL |
30μL |
- --->Zymo Clean
- Sample No.3 -->9μl
- --->Gel Check
|
Sample No.3 |
1 |
Loading Dye |
1 |
dH2O |
4 |
TOTAL |
6 |
- Sample No.3 -> OK ・・・30ng?
|
- --->Ligation
Sample No. |
(1) | (2) | (3) | (4) | (5) |
insert①C0178 |
1 | - | - | 1 | - |
insert②C0170 |
- | 1 | - | - | 1 |
vector③J04500 |
- | - | 2 | 2 | 2 |
ligase |
1 | 1 | 1 | 1 | 1 |
Buffer |
2 | 2 | 2 | 2 | 2 |
dH2O |
6 | 6 | 5 | 4 | 4 |
TOTAL |
10 | 10 | 10 | 10 | 10 |
- --->Transformation
- insert①C0178
- insert②C0170
- vector③J04500
- insert①C0178+vector③J04500
- insert②C0170+vector③J04500
27 August, 2008
Digestion Test
- Sample DNA : BBa_R0010[1]
Sample No. |
1 | 2 | 3 |
Sample DNA |
1 | 1 | 1 |
PstⅠ |
- | - | 0.5 |
EcoRⅠ |
- | 0.5 | 0.5 |
Buffer 3 |
- | 1 | 1 |
BSA |
- | - | 1 |
dH2O |
- | 7.5 | 2 |
TOTAL |
1 | 10 | 10 |
- --->Gel Check
|
Sample No. |
1 | 2 | 3 |
Sample DNA |
1 | 5 | 5 |
Loading Dye |
1 | 1 | 1 |
H2O |
4 | - | - |
TOTAL |
6 | 6 | 6 |
- From left;
- BBa_R0010
- Single Digestion
- Double Digestion
|
- (26/8)--->Colony-PCR
- Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[2](2007)).
DNA Template |
1 |
dNTP mix |
5 |
Foward Primer |
5 |
Reverse Primer |
5 |
DNA polymerase |
0.5 |
Thermopol Buffer |
5 |
dH2O |
28.5 |
TOTAL |
50μL |
- 95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
- --->Gel Check
|
Sample DNA |
10 |
Loading Dye |
2 |
TOTAL |
12 |
- From left;
- 1~5 insert:C0170 + vector:J04500 -> OK
- 6 F2620・・Positive Control -> OK
- 7~11 insert:C0178 + vector:J04500 -> OK
|
- --->(28/8)Miniprep
Transformation
- competent cells : XL10G
-
- --->Mini prep
- --->(29/8)Gel Check
28 August, 2008
- (27/8)--->Mini prep
- insert:C0170 + vector:J04500
- insert:C0178 + vector:J04500
- --->Digestion Test
- insert:C0170 + vector:J04500
- insert:C0178 + vector:J04500
Sample No. |
3 | 4 | 5 | 6 |
insert:C0170 + vector:J04500 |
1 | - | 1 | - |
insert:C0178 + vector:J04500 |
- | 1 | - | 1 |
EcoRⅠ |
0.1 | 0.1 | 0.1 | 0.1 |
SpeⅠ |
- | - | 0.1 | 0.1 |
Buffer 3 |
1 | 1 | 1 | 1 |
BSA |
- | - | 1 | 1 |
dH2O |
7.8 | 7.8 | 6.8 | 6.8 |
TOTAL |
10 | 10 | 10 | 10 |
- --->(29/8)Gel Check
|
Sample No. |
1,2 | 3~6 |
Sample DNA |
1 | 10 |
Loading Dye |
1 | 2 |
H2O |
4 | - |
TOTAL |
6 | 12 |
- From left;
- -> OK
- -> OK
- -> OK
- -> OK
- -> OK
- -> OK
|
29 August, 2008
- --->Digestion Test
- BBa_R0010(2007)
- BBa_C0062(2007)
- BBa_C0179(2007)
- BBa_C0179(2006)
- BBa_S03156(2007)
- BBa_S03156(2006)
- BBa_S03158(2007)
- BBa_S03158(2006)
- BBa_S03160(2007)
- BBa_S03160(2006)
Sample No. |
S1~10 | W1 | W2~10 |
Sample DNA |
1 | 4 | 2 |
PstⅠ |
- | 0.075 | 0.1 |
EcoRⅠ |
0.1 | 0.075 | 0.1 |
Buffer 3 |
1 | 0.75 | 1 |
BSA |
- | 0.75 | 1 |
dH2O |
7.9 | 4.35 | 5.8 |
TOTAL |
10 | 10 | 10 |
- --->Gel Check
|
Sample No. |
1~10 | S1~S10,W1~W10 |
Sample DNA |
1 | 10 |
Loading Dye |
1 | 2 |
TOTAL |
6 | 12 |
- From left;
- 1~5,S1,S5,W1~W5
|
|
- From left;
- 6~10,S6~S10,W6~W10
|
- (27/8)--->Gel Check
|
Sample DNA |
0.5 |
Loading Dye |
1 |
dH2O |
4.5 |
TOTAL |
6μL |
- R0010 -> OK
|
Digestion
- BBa_I9026(2007)
- BBa_I9030(2007)
- BBa_S03154(2007)
- BBa_R0010(2006)
Sample No. |
1~3 | 4 |
Sample DNA |
100ng/μL×20 | 200ng/μL×30 |
PstⅠ |
2 | 2 |
XbaⅠ |
2 | - |
SpeⅠ |
- | 2 |
Buffer 2 |
- | 5 |
Buffer 3 |
5 | - |
BSA |
5 | 5 |
dH2O |
16 | 6 |
TOTAL |
50 | 50 |
- --->Gel Check
|
Sample No. |
1~3 | 4 |
Sample DNA |
10 | 3 |
Loading Dye |
2 | 2 |
dH2O |
- | 7 |
TOTAL |
12 | 12 |
- insert-I9026 : 685bp -> None
|
|
- insert-I9030 : 745bp -> None
|
|
- insert-S03154 : 685bp -> None
|
|
- vector-R0010 : 2079bp -> OK
|
- --->Gel Extract
- --->Zymo Clean
- ->15μL
- --->SAP
Sample No. |
4 |
Sample DNA |
15 |
SAP |
2 |
SAP Buffer |
3 |
dH2O |
10 |
TOTAL |
30μL |
- --->Zymo Clean
- --->Suspension! (We got information, by the team-output, to the effect that the density of the sample is too low.)
30 August, 2008
- Transformation
- competent cells : XL10G
-
- --->(31/8)Mini prep
- (29/8)--->Mini prep
- insert:C0170 + vector:J04500
- insert:C0178 + vector:J04500
- --->Digestion Test
- insert:C0170 + vector:J04500 -> Sample No.1~4
- insert:C0178 + vector:J04500 -> Sample No.5~8
- BBa_T9002(2007) -> Sample No.9
- Single Digestion of Sample No.1~9 -> Sample No.10~18
- Double Digestion of Sample No.1~9 -> Sample No.19~27
Sample No. | Single : 10~18 | Double : 19~27 |
Sample DNA | 1 | 5 |
XbaⅠ | 0.1 | 0.1 |
SpeⅠ | - | 0.1 |
Buffer 2 | 1 | 1 |
BSA | 1 | 1 |
dH2O | 6.9 | 2.8 |
TOTAL | 10 | 10 |
- --->Gel Check
|
Sample No. |
1~9 | 10~18 | 19~27 |
Sample DNA |
1 | 10 | 10 |
Loading Dye |
1 | 2 | 2 |
dH2O |
4 | - | - |
TOTAL |
6 | 12 | 12 |
- From left;
- Sanple No.1~13
- -> OK
|
|
- From left;
- Sample No.14~17,24~16
- 14 -> OK
- 15,16 -> Bad
- 17 -> None
- 24~16 -> Bad
|
|
- From left;
- Sample No.18,27,19~23
- 18,27 -> Bad
- 19~22 -> OK
- 23 -> OK??
|