Team:Kyoto/Notebook
From 2014.igem.org
Revision as of 07:32, 27 September 2014 by ARIATHEiGEM (Talk | contribs)
count down
Home | Team | Official Team Profile | Project | Parts | Modeling | Notebook | Safety | Attributions |
Notebook
Calendar
July | ||||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | ||
6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 | 31 |
August | ||||||
---|---|---|---|---|---|---|
1 | 2 | |||||
3 | 4 | 5 | 6 | 7 | 8 | 9 |
10 | 11 | 12 | 13 | 14 | 15 | 16 |
17 | 18 | 19 | 20 | 21 | 22 | 23 |
24 | 25 | 26 | 27 | 28 | 29 | 30 |
31 |
September | ||||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | |
7 | 8 | 9 | 10 | 11 | 12 | 13 |
14 | 15 | 16 | 17 | 18 | 19 | 20 |
21 | 22 | 23 | 24 | 25 | 26 | 27 |
28 | 29 | 30 |
Octorber | ||||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | |||
5 | 6 | 7 | 8 | 9 | 10 | 11 |
12 | 13 | 14 | 15 | 16 | 17 | 18 |
19 | 20 | 21 | 22 | 23 | 24 | 25 |
26 | 27 | 28 | 29 | 30 | 31 |
7/15
transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_J23101(Pcon) | 2 | 20(2/23) | 22 | LB(CP) |
BBa_B0032(RBS) | 2 | 20(2/23) | 22 | LB(CP) |
7/16
transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_B0030(1) | 2 | 20(2/23) | 22 | LB(CP) |
BBa_B0030(2) | 2 | 20(bought) | 22 | LB(CP) |
BBa_B0032(1) | 2 | 20(2/23) | 22 | LB(CP) |
BBa_B0032(2) | 2 | 20(bought) | 22 | LB(CP) |
7/17
Colony PCR
NameⅠ | NameⅡ |
---|---|
7/16 BBa_B0030(1) Colony_1 | 7/16 BBa_B0032(1) Colony_1 |
7/16 BBa_B0030(1) Colony_2 | 7/16 BBa_B0032(1) Colony_2 |
7/16 BBa_B0030(1) Colony_3 | 7/16 BBa_B0032(1) Colony_3 |
7/16 BBa_B0030(2) Colony_1 | 7/16 BBa_B0032(2) Colony_1 |
7/16 BBa_B0030(2) Colony_2 | 7/16 BBa_B0032(2) Colony_2 |
7/16 BBa_B0030(2) Colony_3 | 7/16 BBa_B0032(2) Colony_3 |
Negative Control | - |
- | - |
Liquid Culture
Name | medium |
---|---|
7/16 BBa_B0030(1) Colony_1 | Plusgrow medium(CP) |
7/16 BBa_B0030(1) Colony_2 | Plusgrow medium(CP) |
7/16 BBa_B0030(1) Colony_3 | Plusgrow medium(CP) |
7/16 BBa_B0030(2) Colony_1 | Plusgrow medium(CP) |
7/16 BBa_B0030(2) Colony_2 | Plusgrow medium(CP) |
7/16 BBa_B0030(2) Colony_3 | Plusgrow medium(CP) |
7/16 BBa_B0032(1) Colony_1 | Plusgrow medium(CP) |
7/16 BBa_B0032(1) Colony_2 | Plusgrow medium(CP) |
7/16 BBa_B0032(1) Colony_3 | Plusgrow medium(CP) |
7/16 BBa_B0032(2) Colony_1 | Plusgrow medium(CP) |
7/16 BBa_B0032(2) Colony_2 | Plusgrow medium(CP) |
7/16 BBa_B0032(2) Colony_3 | Plusgrow medium(CP) |
7/18
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
7/16 BBa_B0030(1) Colony_1 | 229 | 1.96 | 1.40 |
7/16 BBa_B0030(1) Colony_2 | 209 | 1.95 | 1.50 |
7/16 BBa_B0030(1) Colony_3 | 108 | 2.12 | 2.12 |
7/16 BBa_B0030(2) Colony_1 | - | - | - |
7/16 BBa_B0030(2) Colony_2 | 174 | 2.02 | 1.56 |
7/16 BBa_B0030(2) Colony_3 | 158 | 2.01 | 1.68 |
7/16 BBa_B0032(1) Colony_1 | 179 | 2.00 | 2.06 |
7/16 BBa_B0032(1) Colony_2 | - | - | - |
7/16 BBa_B0032(1) Colony_3 | 221 | 1.90 | 1.37 |
7/16 BBa_B0032(2) Colony_1 | 225 | 1.89 | 1.37 |
7/16 BBa_B0032(2) Colony_2 | 270 | 1.98 | 1.63 |
7/16 BBa_B0032(2) Colony_3 | 194 | 1.89 | 1.33 |
7/30
Restriction Enzyme Digestion
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffur/(µl) | BSA/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|---|---|---|
7/16 BBa_B0030(2) Colony_1 | 1 | 0.3 | 0 | 0 | 0 | 1(H) | 0 | 7.7 | 10 |
7/16 BBa_B0030(2) Colony_1 | 1 | 0 | 0.3 | 0 | 0 | 1(M) | 1 | 6.7 | 10 |
7/16 BBa_B0030(2) Colony_1 | 1 | 0 | 0 | 0.3 | 0 | 1(M) | 0 | 7.7 | 10 |
7/16 BBa_B0030(2) Colony_1 | 1 | 0 | 0 | 0 | 0.3 | 1(H) | 0 | 7.7 | 10 |
7/16 BBa_B0030(2) Colony_1(Nega) | 1 | 0 | 0 | 0 | 0 | 1(M) | 0 | 8 | 10 |
Electrophoresis
Lane | Restriction Enzyme Digestion Products |
---|---|
1 | 7/30 BBa_B0030(2) Colony_1(E CUT) |
2 | 7/30 BBa_B0030(2) Colony_1(X CUT) |
3 | 7/30 BBa_B0030(2) Colony_1(S CUT) |
4 | 7/30 BBa_B0030(2) Colony_1(P CUT) |
5 | 7/30 BBa_B0030(2) Colony_1(Nega) |
6 | 1kbp ladder |
7/31
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_E0240(RBS-GFP-DT) | 2 | 20 | 22 | LB(CP) |
J23100(Pcon) | 2 | 20 | 22 | LB(AMP) |
Liquid Culture
Name | medium |
---|---|
7/31 J23100(Pcon) Colony_1 | Plusgrow medium(AMP) |
7/31 J23100(Pcon) Colony_2 | Plusgrow medium(AMP) |
7/31 J23100(Pcon) Colony_3 | Plusgrow medium(AMP) |
7/31 J23100(Pcon) Colony_4 | Plusgrow medium(AMP) |
7/31 J23100(Pcon) Colony_5 | Plusgrow medium(AMP) |
7/31 J23100(Pcon) Colony_6 | Plusgrow medium(AMP) |
Restriction Enzyme Digestion
7/18 RBS Sample | DNA/(µl) | EcoR1/(µl) | Spe1/(µl) | Pst1/(µl) | Xba1/(µl) | Buffur/(µl) | BSA/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|---|---|---|
BBa_B0032 | 5 | 0.3 | 0 | 0 | 0 | 1(H) | 0 | 3.7 | 10 |
BBa_B0032 | 5 | 0 | 0.3 | 0 | 0 | 1(M) | 0 | 3.7 | 10 |
BBa_B0032 | 5 | 0 | 0 | 0.3 | 0 | 1(H) | 0 | 3.7 | 10 |
BBa_B0032 | 5 | 0 | 0 | 0 | 0.3 | 1(M) | 1 | 2.7 | 10 |
BBa_B0032(Nega) | 5 | 0 | 0 | 0 | 0 | 1(M) | 0 | 4 | 10 |
8/1
Electrophoresis
Lane | Restriction Enzyme Digestion Products |
---|---|
1 | 1kb ladder |
2 | 7/31 BBa_B0032(E CUT) |
3 | 7/31 BBa_B0032(S CUT)Error |
4 | - |
5 | - |
6 | 7/31 BBa_B0032(P CUT) |
7 | 7/31 BBa_B0032(X CUT) |
8 | 7/31 BBa_B0032(Nega) |
PCR
BBa_E0240(RBS-GFP-DT) | Volume |
---|---|
10×buffur/(µl) | 5 |
2mMdNTPs/(µl) | 5 |
25mM MgSO4/(µl) | 2 |
VF Primer/(µl) | 1.5 |
VR Primer/(µl) | 1.5 |
template/(µl) | 0.5 |
KOD-plus/(µl) | 1 |
MilliQ/(µl) | 33.5 |
TOTAL/(µl) | 50 |
Steps | Temparature | Time | cycle |
---|---|---|---|
Pre Denature | 94℃ | 2min | 1time |
Denature | 94℃ | 15sec | 30cycle |
Annealing | 55℃ | 30sec | 30cycle |
Extension | 68℃ | 1min | 30cycle |
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
Product | 80 | 1.91 | 1.78 |
8/2
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
7/31 J23100(Pcon) Colony_1 | 145 | 2.11 | 3.09 |
7/31 J23100(Pcon) Colony_2 | - | - | - |
7/31 J23100(Pcon) Colony_3 | 160 | 2.01 | 3.13 |
7/31 J23100(Pcon) Colony_4 | 140 | 1.87 | 2.70 |
7/31 J23100(Pcon) Colony_5 | 105 | 2.39 | 5.90 |
7/31 J23100(Pcon) Colony_6 | 180 | 1.90 | 1.37 |
8/20
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
7/31 BBa_E0240(RBS-GFD-DT) | 2 | 20 | 22 | LB(CP) |
BBa_I732095 | 2 | 20 | 22 | LB(CP) |
8/21
Colony PCR
NameⅠ |
---|
8/20 BBa_I732095 Colony_1 |
8/20 BBa_I732095 Colony_2 |
8/20 BBa_I732095 Colony_3 |
Negative control |
Electrophoresis
Lane | PCR Product |
---|---|
1 | 1kb ladder |
2 | 8/21 BBa_I732095 Colony_1 |
3 | 8/21 BBa_I732095 Colony_2 |
4 | 8/21 BBa_I732095 Colony_3 |
5 | Negative Control |
Liquid Culture
Name | medium |
---|---|
8/20 BBa_I732095 Colony_1 | Plusgrow medium(CP) |
8/20 BBa_I732095 Colony_2 | Plusgrow medium(CP) |
8/20 BBa_I732095 Colony_3 | Plusgrow medium(CP) |
8/22
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/20 BBa_I732095 Colony_1 | 430 | 1.68 | 2.13 |
8/20 BBa_I732095 Colony_2 | 255 | 1.87 | 1.81 |
8/20 BBa_I732095 Colony_3 | 215 | 1.78 | 1.35 |
Restriction Enzyme Digestion
Sample | DNA/(µl) | EcoR1/(µl) | Spe1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|
8/22 BBa_I732095 Colony_1 | 4.7 | 1 | 1 | 3(H) | 20.3 | 30 |
8/22 BBa_I732095 Colony_2 | 7.8 | 1 | 1 | 3(H) | 17.2 | 30 |
8/22 BBa_I732095 Colony_3 | 9.3 | 1 | 1 | 3(H) | 15.7 | 30 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_J04450(pSB3C5) | 2 | 20 | 22 | LB(CP) |
8/25
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_J04450(pSB2K3) | 2 | 20 | 22 | LB(KAN) |
BBa_C0051(pSB1A2) | 2 | 20 | 22 | LB(AMP) |
Liquid Culture
Name | medium |
---|---|
8/22 BBa_J04450(pSB3C5) | Plusgrow medium(CP)*5 |
8/26
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/25 BBa_J04450(pSB3C5) | 575 | 1.37 | 1.37 |
Liquid Culture
Name | medium |
---|---|
8/25 BBa_J04450(pSB2K3) | Plusgrow medium(KAN)*5 |
8/25 BBa_J0051(pSB1A2) | Plusgrow medium(AMP)*2 |
8/27
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/26 BBa_J04450(pSB2K3) | 1045 | 1.43 | 1.11 |
8/26 BBa_J0051(pSB1A2) | 790 | 1.39 | 0.90 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_J04450(pSB3T5) | 2 | 20 | 22 | LB(TET) |
8/28
Liquid Culture
Name | medium |
---|---|
8/27 BBa_J04450(pSB3T5) | Plusgrow medium(TET) |
8/29
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/28 BBa_J04450(pSB3T5) | 180 | 1.60 | 1.67 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
BBa_J04450(pSB4A5) | 2 | 20 | 22 | LB(AMP) |
9/1
Colony PCR
NameⅠ |
---|
- |
- |
- |
9/1 pSB4A5 Colony_1 |
9/1 pSB4A5 Colony_2 |
Negative Control |
Restriction Enzyme Digestion
Sample | DNA/(µl) | EcoR1/(µl) | Pst1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|
8/28 Miniprep pSB3T5 | 25 | 2 | 2 | 6(H) | 25 | 60 |
Negative control_1 | 3.7 | 0 | 0 | 1(H) | 5.3 | 10 |
8/22 Miniprep I732095 | 14 | 3 | 3 | 9(H) | 61 | 90 |
Negative control_2 | 1.5 | 0 | 0 | 1(H) | 7.5 | 10 |
8/27 Miniprep pSB2K3 | 6 | 3 | 3 | 9(H) | 69 | 90 |
Negative control_3 | 1 | 0 | 0 | 1(H) | 8 | 10 |
8/2 Miniprep Pcon Colony_3 | 20 | 1.5 | 1.5 | 5(H) | 17 | 45 |
Negative control_4 | 4 | 0 | 0 | 1(H) | 5 | 10 |
8/2 Miniprep Pcon Colony_1 | 20 | 1.5 | 1.5 | 5(H) | 17 | 45 |
Negative control_5 | 4 | 0 | 0 | 1(H) | 5 | 10 |
Liquid Culture
Name | medium |
---|---|
8/29 BBa_J04450(pSB4A5) Colony_1 | Plusgrow medium(AMP)*2 |
8/1 Pcon Colony_1 | Plusgrow medium(AMP)*2 |
8/27 pSB3T5 Colony_1 | Plusgrow medium(TET)*2 |
9/2
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
8/29 BBa_J04450(pSB4A5) Colony_1 | 210 | 1.61 | 1.18 |
8/1 Pcon Colony_1 | 215 | 1.79 | 1.87 |
8/27 pSB3T5 Colony_1 | 215 | 1.58 | 0.92 |
Gel Extraction
Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
---|---|---|
1 | 9/1 pSB3T5 Colony_1 (Nega) | 10 |
2 | 9/1 pSB3T5 Colony_1 (E+P)(Confirmation) | 5 |
3 | 9/1 pSB3T5 Colony_1 (E+P)(Extraction) | 55 |
4 | 1kb ladder | 3 |
5 | 9/1 I732095 (Nega) | 10 |
6 | 9/1 I732095 (E+P)(Confirmation) | 5 |
7 | 9/1 I732095 (E+P)(Extraction) | 85 |
8 | 1kb ladder | 3 |
9 | 9/1 pSB2K3 (Nega) | 10 |
10 | 9/1 pSB2K3 (E+P)(Confirmation) | 5 |
11 | 9/1 pSB2K3 (E+P)(Extraction) | 85 |
Lane | Sample | Volume/(µl) |
---|---|---|
1 | 9/1 Pcon Colony_3 (E+P)(Confirmation) | 5 |
2 | 9/1 Pcon Colony_3 (E+P)(Extraction) | 20 |
3 | 9/1 Pcon Colony_3 (E+P)(Extraction) | 20 |
4 | 9/1 Pcon Colony_3 (Nega) | 10 |
5 | 1kb ladder | 3 |
6 | 9/1 Pcon Colony_1 (E+P)(Confirmation) | 5 |
7 | 9/1 Pcon Colony_1 (E+P)(Extraction) | 20 |
8 | 9/1 Pcon Colony_1 (E+P)(Extraction) | 20 |
9 | 9/1 Pcon Colony_1 (Nega) | 10 |
9/3
Concentration Measurement
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
pSB2K3 | 40 | 1.48 | 0.10 |
Pcon Colony_1 | 30 | 1.76 | 0.62 |
pSB3T5 | 50 | 2.35 | 0.50 |
pSB1C3 | 65 | 2.14 | 0.10 |
Pcon Colony_3 | 45 | 1.56 | 0.58 |
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
Pcon | 2.1 | pSB1C3 | 1.5 | 1 | 5 | 0.4 | 10 |
-(Negative Control) | 0 | pSB1C3 | 1.5 | 1 | 5 | 2.5 | 10 |
Pcon | 1.3 | pSB2K3 | 2.5 | 1 | 5 | 0.2 | 10 |
-(Negative Control) | 0 | pSB2K3 | 2.5 | 1 | 5 | 1.5 | 10 |
Pcon | 10 | pSB3T5 | 2.3 | 1 | 5 | 0.7 | 10 |
-(Negative Control) | 0 | pSB3T5 | 2.3 | 1 | 5 | 1.7 | 10 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
9/3 Ligation Pcon+pSB1C3 | 3 | 15 | 18 | LB(CP) |
9/3 Ligation pSB1C3(Negative Control) | 8 | 10 | 18 | LB(CP) |
9/3 Ligation Pcon+pSB2K3 | 3 | 15 | 18 | LB(KAN) |
9/3 Ligation pSB2K3(Negative Control) | 8 | 10 | 18 | LB(KAN) |
9/3 Ligation Pcon+pSB3T5 | 3 | 15 | 18 | LB(TET) |
9/3 Ligation pSB3T5(Negative Control) | 2 | 10 | 12 | LB(TET) |
9/4
Colony PCR
NameⅠ | NameⅡ | NameⅢ | NameⅣ |
---|---|---|---|
- | 9/3 pSB1C3(Nega) Colony_7 | 9/3 Pcon+pSB3T5 Colony_5 | 9/3 Pcon+pSB2K3 Colony_1 |
- | 9/3 pSB1C3(Nega) Colony_8 | 9/3 Pcon+pSB3T5 Colony_6 | 9/3 Pcon+pSB2K3 Colony_2 |
9/3 pSB1C3(Nega) Colony_1 | 9/3 Pcon+pSB3T5 Colony_1 | 9/3 Pcon+pSB3T5 Colony_7 | 9/3 Pcon+pSB2K3 Colony_3 |
9/3 pSB1C3(Nega) Colony_2 | 9/3 Pcon+pSB3T5 Colony_2 | 9/3 Pcon+pSB3T5 Colony_8 | 9/3 Pcon+pSB2K3 Colony_4 |
9/3 pSB1C3(Nega) Colony_3 | 9/3 Pcon+pSB3T5 Colony_3 | 9/3 Pcon+pSB3T5 Colony_9 | 9/3 Pcon+pSB2K3 Colony_5 |
9/3 pSB1C3(Nega) Colony_4 | 9/3 Pcon+pSB3T5 Colony_4 | 9/3 Pcon+pSB3T5 Colony_10 | 9/3 Pcon+pSB2K3 Colony_6 |
9/3 pSB1C3(Nega) Colony_5 | 9/3 pSB1C3(Nega) Colony_9 | 9/3 pSB3T5(Nega) Colony_1 | 9/3 Pcon+pSB2K3 Colony_7 |
9/3 pSB1C3(Nega) Colony_6 | 9/3 pSB1C3(Nega) Colony_10 | 9/3 pSB3T5(Nega) Colony_2 | 9/3 Pcon+pSB2K3 Colony_8 |
NameⅤ | NameⅥ | NameⅦ | NameⅧ |
---|---|---|---|
9/3 Pcon+pSB2K3 Colony_9 | 9/3 Pcon+pSB1C3 Colony_2 | 9/3 Pcon+pSB1C3 Colony_10 | 9/3 pSB1C3(Nega) Colony_8 |
9/3 Pcon+pSB2K3 Colony_10 | 9/3 Pcon+pSB1C3 Colony_3 | 9/3 pSB1C3(Nega) Colony_1 | 9/3 pSB1C3(Nega) Colony_9 |
9/3 pSB2K3(Nega) Colony_1 | 9/3 Pcon+pSB1C3 Colony_4 | 9/3 pSB1C3(Nega) Colony_2 | 9/3 pSB1C3(Nega) Colony_10 |
9/3 pSB2K3(Nega) Colony_2 | 9/3 Pcon+pSB1C3 Colony_5 | 9/3 pSB1C3(Nega) Colony_3 | 9/3 pSB1C3(Nega) Colony_11 |
9/3 pSB2K3(Nega) Colony_3 | 9/3 Pcon+pSB1C3 Colony_6 | 9/3 pSB1C3(Nega) Colony_4 | - |
9/3 pSB2K3(Nega) Colony_4 | 9/3 Pcon+pSB1C3 Colony_7 | 9/3 pSB1C3(Nega) Colony_5 | - |
9/3 pSB2K3(Nega) Colony_5 | 9/3 Pcon+pSB1C3 Colony_8 | 9/3 pSB1C3(Nega) Colony_6 | - |
9/3 Pcon+pSB1C3 Colony_1 | 9/3 Pcon+pSB1C3 Colony_9 | 9/3 pSB1C3(Nega) Colony_7 | - |
NameⅨ | NameⅩ |
---|---|
9/3 Pcon+pSB1C3 Colony_11 | 8/22 Miniprep I732095 Colony_3 |
Negative Control | - |
Restriction Enzyme Digestion
Sample | DNA/(µl) | EcoR1/(µl) | Pst1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|
8/27 Miniprep pSB2K3 | 2 | 1 | 1 | 3(H) | 23 | 30 |
8/27 Miniprep pSB2K3(Nega) | 1 | 0 | 0 | 1(H) | 8 | 10 |
9/2 Miniprep pSB3T5 | 10 | 1 | 1 | 3(H) | 15 | 30 |
9/2 Miniprep pSB3T5(Nega) | 3 | 0 | 0 | 1(H) | 6 | 10 |
9/2 Miniprep pSB4A5 | 10 | 1 | 1 | 3(H) | 15 | 30 |
9/2 Miniprep pSB4A5(Nega) | 3 | 0 | 0 | 1(H) | 6 | 10 |
8/22 Miniprep pSB1C3 Colony_2 | 2 | 1 | 1 | 1(H) | 5 | 10 |
9/5
Electrophoresis
Lane | Colony PCR ProductsⅠ | Lane | Colony PCR ProductsⅠ |
---|---|---|---|
1 | 1kb ladder | 10 | 9/4 pSB1C3(Nega) Colony_7 |
2 | - | 11 | 9/4 pSB1C3(Nega) Colony_8 |
3 | - | 12 | 9/4 pSB1C3(Nega) Colony_9 |
4 | 9/4 pSB1C3(Nega) Colony_1 | 13 | 9/4 pSB1C3(Nega) Colony_10 |
5 | 9/4 pSB1C3(Nega) Colony_2 | 14 | 9/4 Pcon+pSB2K3 Colony_1 |
6 | 9/4 pSB1C3(Nega) Colony_3 | 15 | 9/4 Pcon+pSB2K3 Colony_2 |
7 | 9/4 pSB1C3(Nega) Colony_4 | 16 | 9/4 Pcon+pSB2K3 Colony_3 |
8 | 9/4 pSB1C3(Nega) Colony_5 | 17 | 1kb ladder |
9 | 9/4 pSB1C3(Nega) Colony_6 |
Lane | Colony PCR ProductsⅡ | Lane | Colony PCR ProductsⅡ |
---|---|---|---|
1 | 1kb ladder | 10 | 9/4 pSB3T5(Nega) Colony_2 |
2 | 9/4 Pcon+pSB2K3 Colony_4 | 11 | 9/4 Pcon+pSB2K3 Colony_1 |
3 | 9/4 Pcon+pSB2K3 Colony_5 | 12 | 9/4 Pcon+pSB2K3 Colony_2 |
4 | 9/4 Pcon+pSB2K3 Colony_6 | 13 | 9/4 Pcon+pSB2K3 Colony_3 |
5 | 9/4 Pcon+pSB2K3 Colony_7 | 14 | 9/4 Pcon+pSB2K3 Colony_4 |
6 | 9/4 Pcon+pSB2K3 Colony_8 | 15 | 9/4 Pcon+pSB2K3 Colony_5 |
7 | 9/4 Pcon+pSB2K3 Colony_9 | 16 | 9/4 Pcon+pSB2K3 Colony_6 |
8 | 9/4 Pcon+pSB3T5 Colony_10 | 17 | 1kb ladder |
9 | 9/4 pSB3T5(Nega) Colony_1 |
Lane | Colony PCR ProductsⅢ | Lane | Colony PCR ProductsⅢ |
---|---|---|---|
1 | 1kb ladder | 10 | 9/4 pSB2K3(Nega) Colony_5 |
2 | 9/4 Pcon+pSB2K3 Colony_7 | 11 | 9/4 pSB1C3(Nega) Colony_1 |
3 | 9/4 Pcon+pSB2K3 Colony_8 | 12 | 9/4 pSB1C3(Nega) Colony_2 |
4 | 9/4 Pcon+pSB2K3 Colony_9 | 13 | 9/4 pSB1C3(Nega) Colony_3 |
5 | 9/4 Pcon+pSB2K3 Colony_10 | 14 | 9/4 pSB1C3(Nega) Colony_4 |
6 | 9/4 pSB2K3(Nega) Colony_1 | 15 | 9/4 pSB1C3(Nega) Colony_5 |
7 | 9/4 pSB2K3(Nega) Colony_2 | 16 | 9/4 pSB1C3(Nega) Colony_6 |
8 | 9/4 pSB2K3(Nega) Colony_3 | 17 | 1kb ladder |
9 | 9/4 pSB2K3(Nega) Colony_4 |
Lane | Colony PCR ProductsⅣ | Lane | Colony PCR ProductsⅣ |
---|---|---|---|
1 | 1kb ladder | 10 | 9/4 Pcon+pSB1C3 Colony_5 |
2 | 9/4 pSB1C3(Nega) Colony_7 | 11 | 9/4 Pcon+pSB1C3 Colony_6 |
3 | 9/4 pSB1C3(Nega) Colony_8 | 12 | 9/4 Pcon+pSB1C3 Colony_7 |
4 | 9/4 pSB1C3(Nega) Colony_9 | 13 | 9/4 Pcon+pSB1C3 Colony_8 |
5 | 9/4 pSB1C3(Nega) Colony_10 | 14 | 9/4 Pcon+pSB1C3 Colony_9 |
6 | 9/4 Pcon+pSB1C3 Colony_1 | 15 | 9/4 Pcon+pSB1C3 Colony_10 |
7 | 9/4 Pcon+pSB1C3 Colony_2 | 16 | 8/22 Miniprep BBa_I732095 Colony_3 |
8 | 9/4 Pcon+pSB1C3 Colony_3 | 17 | 1kb ladder |
9 | 9/4 Pcon+pSB1C3 Colony_4 |
Lane | Colony PCR ProductsⅤ |
---|---|
1 | 1kb ladder |
2 | 9/4 Pcon+pSB1C3 Colony_11 |
3 | 9/4 Negative Control |
4 | 8/22 Miniprep BBa_I732095 Colony_3 |
5 | - |
6 | - |
7 | - |
8 | - |
Gel Extraction
Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 9/4 pSB2K3(E+P) | 5 |
3 | - | - |
4 | 9/4 pSB2K3(E+P) | 25 |
5 | - | - |
6 | 9/4 pSB2K3(Nega) | 10 |
Lane | Restriction Enzyme Digestion ProductⅡ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 9/4 pSB3T5(E+P) | 5 |
3 | - | - |
4 | 9/4 pSB3T5(E+P) | 25 |
5 | - | - |
6 | 9/4 pSB3T5(Nega) | 10 |
Lane | Restriction Enzyme Digestion ProductⅢ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | 9/4 pSB4A5(E+P) | 5 |
3 | - | - |
4 | 9/4 pSB4A5(E+P) | 25 |
5 | - | - |
6 | 9/4 pSB4A5(Nega) | 10 |
Lane | Restriction Enzyme Digestion ProductⅣ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 5 |
2 | - | - |
3 | - | - |
4 | 9/4 pSB1C3(E+P) | 10 |
5 | - | - |
6 | - | - |
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/4 pSB2K3(E+P) | 36 | 1.25 | 0.24 |
9/4 pSB3T5(E+P) | 10 | 1.00 | -/td> |
9/4 pSB4A5(E+P) | 40 | - | 0.01 |
9/4 pSB1C3(E+P) | 75 | 1.35 | 0.14 |
9/8
Cloning PCR
Target | mamB | mamQ | mamL |
---|---|---|---|
2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 | 12.5 | 12.5 |
Fw Primer/(µl) | 1.5(mamB) | 1.5(mamQ) | 1.5(mamL) |
Rv Primer/(µl) | 1.5(mamB) | 1.5(mamQ) | 1.5(mamL) |
AMB-1 Genome/(µl) | 1 | 1 | 1 |
MilliQ/(µl) | 10 | 10 | 10 |
TOTAL/(µl) | 25 | 25 | 25 |
Steps | Temparature | Time | cycle |
---|---|---|---|
Pre Denature | 98℃ | 5min | 1time |
Denature | 98℃ | 20sec | 25cycle |
Annealing | 65℃ | 15sec | 25cycle |
Extension | 72℃ | 5min | 25cycle |
Final elongation | 72℃ | 5min | 1time |
Final hold | 4℃ | - | - |
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
Pcon | 1 | pSB3T5 | 10 | 1 | 11 | 0 | 22 |
-(Negative Control) | 0 | pSB1C3 | 3 | 0 | 3 | 0 | 6 |
Electrophoresis
Lane | Cloning PCR Products |
---|---|
1 | 1kb ladder |
2 | 9/8 Cloning PCR (AMB-1 mamB) |
3 | 9/8 Cloning PCR (AMB-1 mamQ) |
4 | 9/8 Cloning PCR (AMB-1 mamL) |
5 | 1kb ladder |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
9/8 Ligation Pcon+pSB3T5 | 3 | 30 | 33 | LB(TET) |
9/8 Ligation pSB3T5(Negative Control) | 3 | 30 | 33 | LB(TET) |
Liquid Culture
Name | medium |
---|---|
9/3 Transformation Pcon+pSB1C3 Colony_8 | Plusgrow medium(CP)*2 |
9/9
DNA Purification
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/8 Cloning PCR (AMB-1 mamB) | 165 | 1.54 | 1.57 |
9/8 Cloning PCR (AMB-1 mamQ) | 250 | 1.69 | 1.92 |
9/8 Cloning PCR (AMB-1 mamL) | 190 | 1.56 | 1.79 |
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/8 Liquid Culture Pcon+pSB1C3 | 90 | 1.65 | 1.17 |
Colony PCR
NameⅠ |
---|
- |
9/8 Transformation Pcon+pSB3T5 Colony_1 |
9/8 Transformation Pcon+pSB3T5 Colony_2 |
9/8 Transformation Pcon+pSB3T5 Colony_3 |
9/8 Transformation Pcon+pSB3T5 Colony_4 |
9/8 Transformation Pcon+pSB3T5 Colony_5 |
9/8 Transformation pSB3T5(Nega) Colony_1 |
Negative control |
Electrophoresis
Lane | Cloning PCR Products |
---|---|
1 | 1kb ladder |
2 | 9/8 Colony PCR Pcon+pSB3T5 Colony_1 |
3 | 9/8 Colony PCR Pcon+pSB3T5 Colony_2 |
4 | 9/8 Colony PCR Pcon+pSB3T5 Colony_3 |
5 | 9/8 Colony PCR Pcon+pSB3T5 Colony_4 |
6 | 9/8 Colony PCR Pcon+pSB3T5 Colony_5 |
7 | 9/8 Colony PCR pSB3T5(Nega) Colony_1 |
8 | 9/8 Colony PCR Nega |
Restriction Enzyme Digestion
Sample | DNA/(µl) | Pst1/(µl) | Spe1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|
9/9 Miniprep Pcon+pSB1C3 | 22 | 1 | 1 | 3(H) | 3 | 30 |
9/9 Miniprep Pcon+pSB1C3(Nega) | 1 | 0 | 0 | 1(H) | 8 | 10 |
8/2 Miniprep Pcon Colony_4 | 14 | 1 | 1 | 3(H) | 11 | 30 |
8/2 Miniprep Pcon Colony_4(Nega) | 1 | 0 | 0 | 1(H) | 8 | 10 |
9/10
Gel Extraction
Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | - |
2 | - | - |
3 | 9/10 Digestion Pcon+pSB1C3(S+P) | - |
4 | - | - |
5 | 9/10 Digestion Pcon+pSB1C3(Nega) | - |
6 | - | - |
7 | Pcon(X+P) | - |
8 | - | - |
9 | Pcon(Nega) | - |
10 | 1kb ladder | - |
11 | - | - |
PCR
Target | E+X+RBS+mamB+S | X+RBS+mamL+His+S+P | X+RBS+mamQ+His+S+P |
---|---|---|---|
2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 | 12.5 | 12.5 |
Fw Primer/(µl) | 0.75(E+X+RBS+mamB) | 0.75(X+RBS+mamL) | 0.75(X+RBS+mamQ) |
Rv Primer/(µl) | 0.75(S+mamB) | 0.75(P+S+His+mamL) | 0.75(P+S+His+mamQ) |
9/9 DNA Purification Product/(µl) | Trace | Trace | Trace |
MilliQ/(µl) | 11 | 11 | 11 |
TOTAL/(µl) | 25 | 25 | 25 |
Steps | Temparature | Time | cycle |
---|---|---|---|
Pre Denature | 98℃ | 5min | 1time |
Denature | 98℃ | 20sec | 25cycle |
Annealing | 60℃ | 15sec | 25cycle |
Extension | 72℃ | 15sec | 25cycle |
Final elongation | 72℃ | 5min | 1time |
Final hold | 12℃ | - | - |
Electrophoresis
Lane | Cloning PCR Products |
---|---|
1 | 1kb ladder |
2 | 9/10 PCR E+X+RBS+mamB+S(922bp) |
3 | 9/10 PCR X+RBS+mamL+His+S+P(340bp) |
4 | - |
5 | 1kb ladder |
6 | 9/10 PCR X+RBS+mamQ+His+S+P(953bp) |
DNA Purification
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/10 PCR E+X+RBS+mamB+S | 200 | 1.84 | 2.15 |
9/10 PCR X+RBS+mamL+His+S+P | 120 | 1.69 | 1.84 |
9/10 PCR X+RBS+mamQ+His+S+P | 200 | 1.70 | 2.16 |
Restriction Enzyme Digestion
Sample | DNA/(µl) | Xba1/(µl) | Pst1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|
9/10 PCR E+X+RBS+mamB+S | 10 | 1 | 1 | 3(H) | 15 | 30 |
9/10 PCR X+RBS+mamL+His+S+P | 16.7 | 1 | 1 | 3(H) | 8.3 | 30 |
9/10 PCR X+RBS+mamQ+His+S+P | 10 | 1 | 1 | 3(H) | 15 | 30 |
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
9/10 DNA Purification mamQ | 1 | YCplac111 | 1 | 1 | 5 | 2 | 10 |
9/10 DNA Purification mamL | 1 | YCplac111 | 1 | 1 | 5 | 2 | 10 |
9/10 DNA Purification mamB | 1 | YCplac111 | 1 | 1 | 5 | 2 | 10 |
Control | 0 | YCplac111 | 1 | 1 | 5 | 2 | 9 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
9/10 Ligation mamQ+YCplac111 | - | - | - | - |
9/10 Ligation mamL+YCplac111 | - | - | - | - |
9/10 Ligation mamB+YCplac111 | - | - | - | - |
9/11
DNA Purification
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/10 Digestion mamB(X+P) | 65 | 1.68 | 0.51 |
9/10 Digestion mamL(X+P) | 30 | 1.95 | 0.81 |
9/10 Digestion mamQ(X+P) | 60 | 1.65 | 1.22 |
Electrophoresis
Lane | Colony PCR Products |
---|---|
Lane | Colony PCR Products |
---|---|
Lane | Colony PCR Products |
---|---|
Lane | Colony PCR Products |
---|---|
Lane | Colony PCR Products |
---|---|
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
- | - | - | - | - | - | - | - |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
9/11 Ligation mamL | 2 | 10 | 12 | - |
9/11 Ligation mamQ | 1 | 5 | 6 | - |
Liquid Culture
Name | medium |
---|---|
9/8 Master Pcon+pSB1C3 Colony_3 | Plusgrow medium(CP) |
9/10 Transformation mamL+YCplac111 | Plusgrow medium(AMP) |
9/10 Transformation mamQ+YCplac111 | Plusgrow medium(AMP) |
9/12
Colony PCR
NameⅠ |
---|
Electrophoresis
Lane | Colony PCR Products |
---|---|
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/11 Liquid Culture Pcon+pSB1C3 | 110 | 1.83 | 2.63 |
9/11 Liquid Culture mamL+YCplac111 | 300 | 1.75 | 2.38 |
9/11 Liquid Culture mamQ+YCplac111 | 160 | 1.81 | 2.37 |
Restriction Enzyme Digestion
Sample | DNA/(µl) | Xba1/(µl) | Pst1/(µl) | Spe1/(µl) | Buffur/(µl) | BSA | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|---|---|
9/12 Miniprep mamL+YCplac111 | 6.7 | 1 | 1 | 0 | 3(H) | 3 | 15.3 | 30 |
9/12 Miniprep mamQ+YCplac111 | 12.5 | 1 | 1 | 0 | 3(H) | 3 | 9.5 | 30 |
9/12 Miniprep Pcon+pSB1C3 | 18.1 | 0 | 1 | 1 | 3(H) | 0 | 6.9 | 30 |
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
9/12 Digestion mamB(E+S) | 3 | 9/8 Digestion pSB1C3(E+S) | 1 | 1 | 5 | 0 | 10 |
9/10 mamB | 0.5 | YCplac111 | 1 | 1 | 5 | 2.5 | 10 |
DNA Purification
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
mamB(E+S) | 50 | 1.75 | 2.04 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
9/12 Ligation mamB+pSB1C3 | 1 | 10 | 11 | LB(CP) |
9/12 Ligation mamB+YCplac111 | 1 | 10 | 11 | LB(AMP) |
Colony PCR
NameⅠ | NameⅡ |
---|---|
9/11 Transformation mamL+pSB1A2 Colony_1 | 9/11 Transformation mamL+pSB1A2 Colony_9 |
9/11 Transformation mamL+pSB1A2 Colony_2 | 9/11 Transformation mamL+pSB1A2 Colony_10 |
9/11 Transformation mamL+pSB1A2 Colony_3 | 9/11 Transformation mamQ+pSB1A2 Colony_1 |
9/11 Transformation mamL+pSB1A2 Colony_4 | 9/11 Transformation mamQ+pSB1A2 Colony_2 |
9/11 Transformation mamL+pSB1A2 Colony_5 | 9/11 Transformation mamQ+pSB1A2 Colony_3 |
9/11 Transformation mamL+pSB1A2 Colony_6 | 9/11 Transformation mamQ+pSB1A2 Colony_4 |
9/11 Transformation mamL+pSB1A2 Colony_7 | 9/11 Transformation mamQ+pSB1A2 Colony_5 |
9/11 Transformation mamL+pSB1A2 Colony_8 | 9/11 Transformation mamQ+pSB1A2 Colony_6 |
Electrophoresis
Lane | Colony PCR ProductsⅠ | Lane | Colony PCR ProductsⅠ |
---|---|---|---|
1 | 9/12 mamL+pSB1A2 Colony_1 | 10 | 9/12 mamL+pSB1A2 Colony_9 |
2 | 9/12 mamL+pSB1A2 Colony_2 | 11 | 9/12 mamL+pSB1A2 Colony_10 |
3 | 9/12 mamL+pSB1A2 Colony_3 | 12 | 9/12 mamQ+pSB1A2 Colony_1 |
4 | 9/12 mamL+pSB1A2 Colony_4 | 13 | 9/12 mamQ+pSB1A2 Colony_2 |
5 | 9/12 mamL+pSB1A2 Colony_5 | 14 | 9/12 mamQ+pSB1A2 Colony_3 |
6 | 9/12 mamL+pSB1A2 Colony_6 | 15 | 9/12 mamQ+pSB1A2 Colony_4 |
7 | 9/12 mamL+pSB1A2 Colony_7 | 16 | 9/12 mamQ+pSB1A2 Colony_5 |
8 | 9/12 mamL+pSB1A2 Colony_8 | 17 | 9/12 mamQ+pSB1A2 Colony_6 |
9 | 1kb ladder |
Colony PCR
NameⅠ |
---|
9/11 Transformation mamL+pSB1A2 Green_1 |
9/11 Transformation mamL+pSB1A2 Green_2 |
9/11 Transformation mamL+pSB1A2 Green_3 |
9/11 Transformation mamL+pSB1A2 Green_4 |
9/11 Transformation mamL+pSB1A2 Green_5 |
9/11 Transformation mamL+pSB1A2 Black_7 |
9/11 Transformation mamL+pSB1A2 Black_8 |
9/11 Transformation mamQ+pSB1A2 Black_1 |
Electrophoresis
Lane | Colony PCR ProductsⅠ |
---|---|
1 | 9/12 mamL+pSB1A2 Green_1 |
2 | 9/12 mamL+pSB1A2 Green_2 |
3 | 9/12 mamL+pSB1A2 Green_3 |
4 | 9/12 mamL+pSB1A2 Green_4 |
5 | 9/12 mamL+pSB1A2 Green_5 |
6 | 9/12 mamL+pSB1A2 Black_7 |
7 | 9/12 mamL+pSB1A2 Black_8 |
8 | 9/12 mamQ+pSB1A2 Black_1 |
9 | 1kb ladder |
Liquid Culture
Name | medium |
---|---|
9/11 Transformation mamQ+pSB1A2 Colony_1 | Plusgrow medium(AMP) |
9/11 Transformation mamQ+pSB1A2 Colony_2 | Plusgrow medium(AMP) |
9/11 Transformation mamQ+pSB1A2 Colony_3 | Plusgrow medium(AMP) |
9/11 Transformation mamQ+pSB1A2 Colony_4 | Plusgrow medium(AMP) |
9/11 Transformation mamQ+pSB1A2 Colony_5 | Plusgrow medium(AMP) |
9/11 Transformation mamQ+pSB1A2 Colony_6 | Plusgrow medium(AMP) |
9/13
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/12 Liquid Culture mamQ+pSB1A2 Colony_1 Error | - | - | - |
9/12 Liquid Culture mamQ+pSB1A2 Colony_2 Error | - | - | - |
9/12 Liquid Culture mamQ+pSB1A2 Colony_3 Error | - | - | - |
9/12 Liquid Culture mamQ+pSB1A2 Colony_4 | 80 | 2.02 | 2.76 |
9/12 Liquid Culture mamQ+pSB1A2 Colony_5 | 70 | 2.13 | 3.41 |
9/12 Liquid Culture mamQ+pSB1A2 Colony_6 | 110 | 1.96 | 1.70 |
9/12 Liquid Culture Pcon+pSB1C3 | 110 | 1.96 | 1.70 |
PCR Labeling and Sequence
Target | 9/13 mamQ+pSB1A2 Colony_4 | 9/13 mamQ+pSB1A2 Colony_5 | 9/13 mamQ+pSB1A2 Colony_6 | Pcon+pSB1A2 | 9/13 mamQ+pSB1A2 Colony_4 | 9/13 mamQ+pSB1A2 Colony_5 | 9/13 mamQ+pSB1A2 Colony_6 |
---|---|---|---|---|---|---|---|
Template/(µl) | 5 | 4 | 6 | 4 | 5 | 6 | 4 |
Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 |
Primer/(µl) | 0.5(VF) | 0.5(VF) | 0.5(VF) | 0.5(VF) | 0.5(VR) | 0.5(VR) | 0.5(VR) |
MilliQ/(µl) | 2.75 | 1.75 | 3.75 | 3.75 | 2.75 | 1.75 | 3.75 |
TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 |
Target | 9/12 mamQ+YCplac111 | 9/12 mamL+YCplac111 | 9/12 mamQ+YCplac111 | 9/12 mamL+YCplac111 | 9/12 mamQ+YCplac111 |
---|---|---|---|---|---|
Template/(µl) | 3 | 1.5 | 3 | 1.5 | 3 |
Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 |
Primer/(µl) | 0.5(M13F) | 0.5(M13F) | 0.5(M13R) | 0.5(M13R) | 0.5(seqpLQB-2) |
MilliQ/(µl) | 4.75 | 6.25 | 4.75 | 6.25 | 4.75 |
TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 |
Gel Extraction
Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | - |
2 | - | - |
3 | 9/12 Digestion mamQ+YCpla111(X+P)(992bp) | - |
4 | - | - |
5 | - | - |
6 | - | - |
7 | 9/12 Digestion mamL+YCpla111(X+P)(340bp) | - |
8 | - | - |
9 | 9/12 Digestion Pcon+pSB1C3(Nega)(5600bp) | - |
10 | - | - |
11 | 9/12 Digestion Pcon+pSB1C3(S+P)(2070bp) | - |
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/12 Digestion mamL+YCpla111(X+P) | 10 | 1.00 | 0.70 |
9/12 Digestion Pcon+pSB1C3(S+P) | 150 | 1.25 | 0.71 |
Restriction Enzyme Digestion
Sample | DNA/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | EcoR1/(µl) | Buffur/(µl) | BSA | MilliQ/(µl) | TOTAL/(µl) |
---|---|---|---|---|---|---|---|---|---|
9/13 Miniprep mamQ+pSB1A2 Colony_4 | 12.5 | 0.5 | 0 | 0.5 | 0 | 1.7(M) | 1.6 | 0 | 16.8 |
9/13 Miniprep mamQ+pSB1A2 Colony_5 | 14.3 | 0.5 | 0 | 0.5 | 0 | 1.9(M) | 1.86 | 0 | 19.06 |
9/13 Miniprep mamQ+pSB1A2 Colony_6 | 9.1 | 0.5 | 0 | 0.5 | 0 | 1.5(M) | 1.5 | 1.9 | 15 |
9/13 Miniprep mamQ+pSB1A2 Colony_6(Nega) | 3 | 0 | 0 | 0 | 0 | 0.5(M) | 0.5 | 1 | 5 |
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
9/13 Gel Extraction mamL(X+P) | 3 | Pcon+pSB1C3(S+P) | 1 | 1 | 5 | 0 | 10 |
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
9/12 Liquid Culture Pcon+pSB1C3 | 240 | 1.78 | 1.59 |
9/14
Gel Extraction
Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
---|---|---|
1 | 1kb ladder | 3 |
2 | - | - |
3 | 9/13 mamQ Colony_4(X+P) | 16.8 |
4 | - | - |
5 | 1kb ladder | 3 |
6 | - | - |
7 | 9/13 mamQ Colony_5(X+P) | 19.06 |
8 | - | - |
9 | 9/13 mamQ Colony_6(Nega) | 5 |
10 | - | - |
11 | 9/13 mamQ Colony_6(X+P) | 15 |
Ligation
Insert/(µL) | Vector/(µL) | T4 Ligase/(µl) | 2*Buffer/(µl) | MilliQ/(µl) | Total/(µl) | ||
---|---|---|---|---|---|---|---|
mamB(E+S) | 4 | pSB1C3(E+S) | 3 | 1 | 8 | 0 | 16 |
mamL(X+P) | 3.3 | pSB1C3(E+S) | 0.7 | 1 | 5 | 0 | 10 |
PCR
Target | E+X+RBS+mamB+S |
---|---|
2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 |
Fw Primer/(µl) | 0.75(E+X+RBS+mamB) | /tr>
Rv Primer/(µl) | 0.75(S+mamB) |
9/9 DNA Purification Product/(µl) | Trace |
MilliQ/(µl) | 11 |
TOTAL/(µl) | 25 |
Steps | Temparature | Time | cycle |
---|---|---|---|
Pre Denature | 98℃ | 5min | 1time |
Denature | 98℃ | 20sec | 25cycle |
Annealing | 60℃ | 15sec | 25cycle |
Extension | 72℃ | 15sec | 25cycle |
Final elongation | 72℃ | 5min | 1time |
Final hold | 12℃ | - | - |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
9/14 Ligation Pcon+mamL+pSB1C3 | 1 | 10 | 11 | LB(CP) |
9/14 Ligation Pcon+mamL+pSB1C3(Nega) | 1 | 10 | 11 | LB(CP) |
9/14 Ligation mamB+pSB1C3 | 1 | 10 | 11 | LB(CP) |
9/15
Colony PCR
NameⅠ | NameⅡ |
---|---|
9/14 Transformation mamB+pSB1C3 Colony_1 | 9/14 Transformation mamB+pSB1C3 Colony_9 |
9/14 Transformation mamB+pSB1C3 Colony_2 | 9/14 Transformation mamB+pSB1C3 Colony_10 |
9/14 Transformation mamB+pSB1C3 Colony_3 | 9/14 Transformation mamB+pSB1C3 Colony_11 |
9/14 Transformation mamB+pSB1C3 Colony_4 | 9/14 Transformation mamB+pSB1C3 Colony_12 |
9/14 Transformation mamB+pSB1C3 Colony_5 | 9/14 Transformation mamB+pSB1C3 Colony_13 |
9/14 Transformation mamB+pSB1C3 Colony_6 | 9/14 Transformation mamB+pSB1C3 Colony_14 |
9/14 Transformation mamB+pSB1C3 Colony_7 | 9/14 Transformation mamB+pSB1C3 Colony_15 |
9/14 Transformation mamB+pSB1C3 Colony_8 | - |
Electrophoresis
Lane | Colony PCR ProductsⅠ |
---|---|
1 | 9/14 mamB+pSB1C3 Colony_1 |
2 | 9/14 mamB+pSB1C3 Colony_2 |
3 | 9/14 mamB+pSB1C3 Colony_3 |
4 | 9/14 mamB+pSB1C3 Colony_4 |
5 | 9/14 mamB+pSB1C3 Colony_5 |
6 | 9/14 mamB+pSB1C3 Colony_6 |
7 | 9/14 mamB+pSB1C3 Colony_7 |
8 | 9/14 mamB+pSB1C3 Colony_8 |
9 | 1kb ladder |
10 | 9/14 mamB+pSB1C3 Colony_9 |
11 | 9/14 mamB+pSB1C3 Colony_10 |
12 | 9/14 mamB+pSB1C3 Colony_11 |
13 | 9/14 mamB+pSB1C3 Colony_12 |
14 | 9/14 mamB+pSB1C3 Colony_13 |
15 | 9/14 mamB+pSB1C3 Colony_14 |
16 | 9/14 mamB+pSB1C3 Colony_15 |
Colony PCR
NameⅠ |
---|
9/14 Transformation Pcon+mamL+SB1C3 Colony_1 |
9/14 Transformation mamB+pSB1C3 Colony_9 |
Transformation
Sample/(µL) | CompetentCells/(µl) | Total/(µl) | Medium | |
---|---|---|---|---|
mamB+YCplac111 | 1 | 10 | 11 | LB(AMP) |
Pcon+mamL+pSB1C3 | 1 | 10 | 11 | LB(CP) |
Miniprep
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
---|---|---|---|
mamB+pSB1C3 Colony_9 | - | - | - |
mamB+pSB1C3 Colony_10 | - | - | - |
mamB+pSB1C3 Colony_11 | - | - | - |
mamL+pSB1A2 Colony_1 | 170 | 1.74 | 1.41 |
mamL+pSB1A2 Colony_2 | 140 | 1.95 | 1.43 |
mamL+pSB1A2 Colony_3 | 135 | 1.95 | 1.60 |
mamL+pSB1A2 Colony_4 | - | - | - |
mamL+pSB1A2 Colony_5 | - | - | - |
mamL+pSB1A2 Colony_6 | - | - | - |