Team:Hong Kong-CUHK/Notebook
From 2011.igem.org
Line 12,527: | Line 12,527: | ||
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Lane6&7: T7HR1C3 cut by Ecor1 and Pst1</b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Lane6&7: T7HR1C3 cut by Ecor1 and Pst1</b></div> | ||
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Results: correct size of T7HR24C1C3 <span>biobricks Successfully inserted the 24C double terminator.</span></b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Results: correct size of T7HR24C1C3 <span>biobricks Successfully inserted the 24C double terminator.</span></b></div> | ||
- | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="516" height="784" v:shapes="_x0000_i1041" alt="" src=" | + | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="516" height="784" v:shapes="_x0000_i1041" alt="" src="http://samlo.t0.ru/igem/stock/image030.jpg" /></b></div> |
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | ||
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><u>23 Sept (Fri)</u></b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><u>23 Sept (Fri)</u></b></div> | ||
Line 12,560: | Line 12,560: | ||
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Lane 5: HR1C3 cut by EcoR1 and Pst1</b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Lane 5: HR1C3 cut by EcoR1 and Pst1</b></div> | ||
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | ||
- | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="311" height="307" v:shapes="_x0000_i1042" alt="" src=" | + | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="311" height="307" v:shapes="_x0000_i1042" alt="" src="http://samlo.t0.ru/igem/stock/image032.jpg" /></b></div> |
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | ||
<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> | <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b> </b></div> |
Revision as of 17:53, 5 October 2011
❢ The notebook needs time to load from scratch due to the enormous size of the notebook; please be patient if nothing showed up or the browser is loading, thank you...
Solution (added by order)
|
Amount(ul)
|
ddH20
|
14
|
10X Buffer 3
|
2.5
|
10X BSA
|
2.5
|
EcoR1
|
0.5
|
Pst1
|
0.5
|
HR
|
5
|
Total
|
25ul
|
Solution (added by order)
|
Amount(ul)
|
ddH20
|
14
|
10X Buffer 3
|
2.5
|
10X BSA
|
2.5
|
EcoR1
|
0.5
|
Pst1
|
0.5
|
T7
|
5
|
Total
|
25ul
|
Solution (added by order)
|
Amount(ul)
|
ddH20
|
15
|
10X Buffer 3
|
2.5
|
10X BSA
|
2.5
|
EcoR1
|
0.5
|
Pst1
|
0.5
|
pSB1
|
4
|
Total
|
25ul
|
[Salt]/M
|
0
|
0.1
|
0.2
|
0.3
|
0.4
|
0.5
|
0.6
|
0.7
|
0.8
|
0.9
|
1.0
|
LB + salt/ml
|
0
|
0.4
|
0.8
|
1.2
|
1.6
|
2.0
|
2.4
|
2.8
|
3.2
|
3.6
|
4
|
LB/ml
|
4.0
|
3.6
|
3.2
|
2.8
|
2.4
|
2.0
|
1.6
|
1.2
|
0.8
|
0.4
|
0
|
Light absorbance of DH5α KCl IPTG
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(5x)
|
0.532
|
2.660
|
0.2(5x)
|
0.515
|
2.575
|
0.3(5x)
|
0.477
|
2.385
|
0.4(5x)
|
0.390
|
1.950
|
0.5(5x)
|
0.370
|
1.850
|
0.6(5x)
|
0.267
|
1.335
|
0.7(5x)
|
0.92
|
0.960
|
0.8(2x)
|
0.300
|
0.600
|
0.9(2x)
|
0.156
|
0.312
|
1.0
|
0.249
|
0.249
|
0
|
0.791
|
0.791
|
Light absorbance of DH5α KCl
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(5x)
|
0.500
|
2.500
|
0.2(5x)
|
0.447
|
2.235
|
0.3(5x)
|
0.430
|
2.150
|
0.4(5x)
|
0.399
|
1.995
|
0.5(5x)
|
0.305
|
1.525
|
0.6(5x)
|
0.264
|
1.32
|
0.7(5x)
|
0.205
|
1.025
|
0.8(5x)
|
0.117
|
0.585
|
0.9(2x)
|
0.220
|
0.440
|
1.0
|
0.263
|
0.263
|
0(5x)
|
0.519
|
2.595
|
Light absorbance of DH5α NaCl IPTG
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(2x)
|
0.829
|
1.658
|
0.2(2x)
|
0.844
|
1.688
|
0.3(5x)
|
0.898
|
1.796
|
0.4(5x)
|
0.384
|
1.920
|
0.5(5x)
|
0.340
|
1.700
|
0.6(5x)
|
0.208
|
1.040
|
0.7(2x)
|
0.393
|
0.786
|
0.8(5x)
|
0.287
|
0.574
|
0.9(2x)
|
0.119
|
0.238
|
1.0
|
0.113
|
0.113
|
Light absorbance of DH5α NaCl
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(5x)
|
0.499
|
2.495
|
0.2(2x)
|
0.478
|
2.390
|
0.3(5x)
|
0.513
|
2.565
|
0.4(5x)
|
0.390
|
1.950
|
0.5(5x)
|
0.302
|
1.510
|
0.6(5x)
|
0.246
|
1.230
|
0.7(5x)
|
0.194
|
0.970
|
0.8(2x)
|
0.269
|
0.538
|
0.9
|
0.284
|
0.284
|
1.0
|
0.103
|
0.103
|
BL21
|
0.5ml
|
1.891
|
BL21
|
1.0ml
|
1.831
|
DH5α
|
0.5ml
|
0.670
|
DH5α
|
1.0ml
|
1.415
|
FLASK1: 0.5ml culture + 125LB
|
FLASK2: 0.25 ml culture + 125LB
|
Light absorbance of DH5α KCl IPTG
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(2x)
|
0.916
|
1.832
|
0.2(2x)
|
0.756
|
1.512
|
0.3(2x)
|
0.527
|
1.054
|
0.4
|
0.426
|
0.426
|
0.5
|
0.710
|
0.710
|
0.6
|
0.310
|
0.310
|
0.7
|
0.156
|
0.156
|
0.8
|
0.129
|
0.129
|
0.9
|
0.072
|
0.072
|
1.0
|
0.012
|
0.012
|
Light absorbance of DH5α KCl
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1
|
0.618
|
2.472
|
0.2
|
0.427
|
1.708
|
0.3
|
0.274
|
1.096
|
0.4
|
0.845
|
1.690
|
0.5
|
0.474
|
0.948
|
0.6
|
0.970
|
0.970
|
0.7
|
0.626
|
1.252
|
0.8
|
0.504
|
0.504
|
0.9
|
0.083
|
0.083
|
1.0
|
-0.011
|
-0.011
|
Light absorbance of DH5α NaCl IPTG
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(2x)
|
0.794
|
1.588
|
0.2(2x)
|
0.716
|
1.432
|
0.3(2x)
|
0.483
|
0.966
|
0.4(2x)
|
0.436
|
0.872
|
0.5
|
0.761
|
0.761
|
0.6
|
0.713
|
0.713
|
0.7
|
0.641
|
0.641
|
0.8
|
0.491
|
0.491
|
0.9
|
0.168
|
0.168
|
1.0
|
0.044
|
0.044
|
Light absorbance of DH5α NaCl
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1(2x)
|
0.932
|
1.864
|
0.2(2x)
|
0.719
|
1.438
|
0.3(2x)
|
0.535
|
1.070
|
0.4(2x)
|
0.525
|
1.050
|
0.5
|
0.882
|
0.882
|
0.6
|
0.819
|
0.819
|
0.7
|
0.712
|
0.712
|
0.8
|
0.470
|
0.470
|
0.9
|
0.123
|
0.123
|
1.0
|
0.031
|
0.031
|
Light absorbance of DH5α 0M LB
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1
|
0.763
|
1.526
|
0.2
|
0.829
|
1.658
|
Time(h)\OD of Solution
|
LB
|
LB IPTG
|
0.6M NaCl LB
|
0.6M NaCl LB IPTG
|
0.6M KCl LB
|
0.6M KCl LB IPTG
|
0
|
0.015
|
0.016
|
0.028
|
0.030
|
0.025
|
0.047
|
0.5
|
0.025
|
0.024
|
0.050
|
0.017
|
0.041
|
0.014
|
1
|
0.035
|
0.039
|
0.077
|
0.015
|
0.025
|
0.029
|
1.5
|
0.071
|
0.080
|
0.070
|
0.005
|
0.036
|
0.026
|
2
|
0.146
|
0.176
|
0.111
|
0.005
|
0.044
|
0.044
|
2.5
|
0.307
|
0.338
|
0.148
|
0.003
|
0.063
|
0.057
|
3
|
0.473
|
0.527
|
0.262
|
0.017
|
0.093
|
0.093
|
3.5
|
0.650
|
0.669
|
0.345
|
0.012
|
0.134
|
0.137
|
4
|
0.874
|
0.888
|
0.473
|
0.003
|
0.219
|
0.210
|
4.5
|
10.66
|
1.128
|
0.626
|
0.001
|
0.323
|
0.310
|
5
|
0.753(2x)
|
0.800(2x)
|
0.722
|
0.001
|
0.386
|
0.365
|
Flask
|
1
|
2
|
3
|
4
|
OD 600
|
1.697
|
1.844
|
1.840
|
1.662
|
|
260/280
|
DNA concentration (ng/ul)
|
T7 pSB1K3 (1)
|
1.95
|
32.6
|
T7 pSB1K3 (2)
|
1.90
|
32.2
|
HR pSB1K3 (1)
|
2.08
|
12.2
|
HR pSB1K3 (2)
|
2.01
|
12.3
|
Water
|
11.75ul
|
Buffer 3
|
3.5ul
|
BSA
|
0.35ul
|
EcoR1
|
0.7ul
|
Pst1
|
0.7ul
|
T7 pSB1K3
|
8ul
|
Total
|
25ul
|
Buffer 3
|
2.5ul
|
BSA
|
0.25ul
|
EcoR1
|
0.5ul
|
Pst1
|
0.5ul
|
HR pSB1K3
|
21.25ul
|
Total
|
25ul
|
Time(h)\OD of Solution
|
LB
|
LB IPTG
|
0.6M NaCl LB
|
0.6M NaCl LB IPTG
|
0.6M KCl LB
|
0.6M KCl LB IPTG
|
0
|
0.002
|
0.023
|
0.019
|
0.026
|
0.001
|
0.001
|
0.5
|
0.006
|
0.004
|
0.020
|
0.032
|
0.003
|
0.018
|
1
|
0.012
|
0.022
|
0.043
|
0.031
|
0.011
|
0.006
|
1.5
|
0.023
|
0.028
|
0.047
|
0.021
|
0.017
|
0.032
|
2
|
0.110
|
0.151
|
0.065
|
0.068
|
0.025
|
0.021
|
2.5
|
0.279
|
0.345
|
0.129
|
0.075
|
0.029
|
0.053
|
3
|
0.430
|
0.455
|
0.488
|
0.137
|
0.068
|
0.057
|
3.5
|
0.591.
|
0.640
|
0.230
|
0.183
|
0.089
|
0.112
|
4
|
0.748
|
0.830
|
0.310
|
0.264
|
0.122
|
0.150
|
4.5
|
0.962
|
1.053
|
0.445
|
0.353
|
0.183
|
0.265
|
5
|
0.747
|
0.832
|
0.389
|
0.310
|
0.177
|
0.209
|
5.5
|
0.939
|
10.17
|
0.448
|
0.380
|
0.235
|
0.261
|
6
|
0.764
|
0.745
|
0.360
|
0.316
|
0.251
|
0.310
|
|
Volume (ul)
|
OD 600
|
DH5a
|
50
|
0.352
|
|
100
|
0.523
|
BL 21
|
50
|
0.349
|
|
100
|
1.710
|
Water
|
11.2ul
|
DNA
|
10ul
|
Buffer 3
|
2.5ul
|
EcoR1
|
0.5ul
|
Pst1
|
0.5ul
|
BSA
|
0.25ul
|
Total
|
25ul
|
Well
|
1
|
2
|
3
|
4
|
5
|
6
|
|
ladder
|
T71K3 (1)
|
T71K3 (2)
|
HR(1)
|
HR(2)
|
HR(3)
|
Well
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
|
ladder
|
T7(1)
|
T7(2)
|
T7(3)
|
HR1K3(1)
|
HR1K3(2)
|
HR1K3(3)
|
|
OD
|
BL21 (1)
|
1.958
|
BL21 (2)
|
1.918
|
BL21 (3)
|
1.907
|
Light absorbance of DH5α NaCl IPTG( 4times diluted)
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1
|
0.701
|
2.804
|
0.2
|
0.786
|
3.144
|
0.3
|
0.680
|
2.720
|
0.4(2x)
|
0.947
|
3.788
|
0.5(2x)
|
0.603
|
2.412
|
0.6(2x)
|
0.538
|
2.152
|
0.7
|
0.334
|
1.336
|
0.8
|
0.083
|
0.332
|
0.9
|
0.025
|
0.100
|
1.0
|
0.022
|
0.088
|
Light absorbance of DH5α KCl IPTG( 4times diluted)
|
||
Concentration/M
|
OD (ABS/cm)
|
Final OD (ABS/cm) (without dilution)
|
0.1
|
0.893
|
3.572
|
0.2
|
0.906
|
3.624
|
0.3
|
0.780
|
3.120
|
0.4
|
0.445
|
1.780
|
0.5
|
0.621
|
2.484
|
0.6
|
0.083
|
0.332
|
0.7
|
0.218
|
0.872
|
0.8
|
0.040
|
0.160
|
0.9
|
0.010
|
0.040
|
1.0
|
0.027
|
0.108
|
Time(h)\OD of Solution
|
LB IPTG
|
0.6M NaCl LB
|
0.6M KCl LB
|
0
|
0.025
|
0.070
|
0.054
|
0.5
|
0.043
|
0.065
|
0.044
|
1
|
0.117
|
0.068
|
0.067
|
1.5
|
0.185
|
0.041
|
0.059
|
2
|
0.389
|
0.069
|
0.064
|
2.5
|
0.634
|
0.085
|
0.081
|
3
|
1.039
|
0.107
|
0.111
|
3.5
|
1.586
|
0.188
|
0.181
|
4
|
0.334
|
0.274
|
0.268
|
4.5
|
0.364
|
0.350
|
0.328
|
5
|
2.412
|
0.432
|
0.450
|
Time(h)
|
Averaged cell density
|
0
|
1.5 x 104
|
0.5
|
2.0 x 104
|
1
|
1.3 x 105
|
1.5
|
2.0 x 106
|
2
|
-
|
2.5
|
-
|
3
|
8.95 x 106
|
3.5
|
8.30 x 109
|
4
|
1.145 x 1010
|
4.5
|
6.70 x 109
|
5
|
6.10 x 109
|
BL21
|
OD
|
1
|
1.094
|
2
|
1.263
|
3
|
1.263
|
4
|
1.163
|
Buffer 3
|
5ul
|
BSA
|
0.5ul
|
Eco R1
|
1ul
|
Pst 1
|
1ul
|
T71K3
|
10ul
|
1. Pick colony of BL21 X4 and inoculation
PCR supermix
|
45ul
|
Upper primer
|
1ul
|
Lower primer
|
1ul
|
Water
|
25ul
|
DNA
|
0.5ul
|
TOTAL
|
50ul
|
|
1
|
2
|
3
|
4
|
16:30
|
0.006
|
0.007
|
0.007
|
0.020
|
16:45
|
0.007
|
0.013
|
0.01
|
0.007
|
20:45
|
0.143
|
0.085
|
0.085
|
0.136
|
22:15
|
0.367
|
|
0.312
|
|
Time(h)\OD600 of Solution
|
LB IPTG
|
0.4M NaCl LB
|
0.4M KCl LB
|
0
|
0.033
|
0.031
|
0.025
|
0.5
|
0.037
|
0.032
|
0.017
|
1
|
0.078
|
0.013
|
0.033
|
1.5
|
0.122
|
0.056
|
0.047
|
2
|
0.310
|
0.102
|
0.096
|
2.5
|
0.423
|
0.157
|
0.156
|
3
|
0.620
|
0.255
|
0.256
|
3.5
|
0.893
|
0.398
|
0.427
|
4
|
1.186
|
0.590
|
0.621
|
4.5
|
1.632
|
0.859
|
0.803
|
Time(h)
|
Averaged cell density
|
0
|
3x106
|
0.5
|
2.5 x106
|
1
|
3.75 x107
|
1.5
|
1 x107
|
2
|
9.75 x108
|
2.5
|
9.15 x108
|
3
|
2.43 x109
|
3.5
|
2.555 x109
|
4
|
2.7875 x109
|
4.5
|
2.225 x109
|
1. iGEM plasmid stock check:
pSB1K3
|
~ 0ul
|
pSB1C3
|
< 1ul
|
pSB1T3
|
~4-5ul
|
pSB1A3
|
~4-5ul
|
1. Nano drop
T7 + 1K3
|
42.5 ng/ul
|
HR + 1K3
|
13.5 ng/ul
|
H2O
|
19.25ul
|
Buffer 2
|
2ul
|
BSA
|
2.5ul
|
Eco R1
|
0.5ul
|
Spe 1
|
0.5ul
|
T71K3
|
0.25ul
|
TOTAL
|
25ul
|
H2O
|
14.25ul
|
Buffer 2
|
2.5ul
|
BSA
|
0.25ul
|
Xbal 1
|
0.5ul
|
Pst 1
|
0.5ul
|
HR1K3
|
7ul
|
TOTAL
|
25ul
|
H2O
|
17.25ul
|
Buffer 2
|
2.5ul
|
BSA
|
0.25ul
|
Eco R1
|
0.5ul
|
Pst 1
|
0.5ul
|
pSB1T3
|
4ul
|
TOTAL
|
25ul
|
Time(h)\OD600 of Solution
|
LB IPTG
|
0.4M NaCl LB
|
0.4M KCl LB
|
0
|
0.180
|
0.049
|
0.025
|
0.5
|
0.234
|
0.021
|
0.020
|
1
|
0.301
|
0.041
|
0.024
|
1.5
|
0.377
|
0.047
|
0.055
|
2
|
0.606
|
0.127
|
0.114
|
2.5
|
0.744
|
0.209
|
0.212
|
3
|
0.916
|
0.324
|
0.331
|
3.5
|
1.125
|
0.472
|
0.449
|
4
|
1.289
|
0.673
|
0.725
|
4.5
|
1.432
|
0.881
|
0.886
|
5
|
1.533
|
10147
|
1.122
|
5.5
|
1.654
|
1.345
|
1.364
|
Time(h)
|
Averaged cell density
|
0
|
2.21x107
|
0.5
|
8.3 x108
|
1
|
7.9 x107
|
1.5
|
3.6 x108
|
2
|
3.035 x109
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
Eco R1
|
0.5ul
|
Spe 1
|
0.5ul
|
T71K3
|
16.8ul
|
TOTAL
|
20ul
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
Xbal 1
|
0.5ul
|
Pst 1
|
0.5ul
|
HR1K3
|
16.8ul
|
TOTAL
|
20ul
|
H2O
|
12.8ul
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
Eco R1
|
0.5ul
|
Pst 1
|
0.5ul
|
pSB1T3
|
4ul
|
TOTAL
|
20ul
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
blank
|
HR+1K3
|
Ladder
|
T7+1K3
|
blank
|
pSB1T3
|
blank
|
blank
|
T4 ligase buffer
|
2ul
|
T4 ligase
|
1ul
|
T7
|
8ul
|
HR
|
6ul
|
Vector (pSB1T3)
|
3ul
|
TOTAL
|
20ul
|
DNA
|
10ul
|
10X Buffer 3
|
2.5ul
|
10X BSA
|
2.5ul
|
EcoR1
|
0.6ul
|
Pst1
|
0.6ul
|
H2O
|
8.8
|
Total
|
25ul
|
Water
|
11.25ul
|
DNA
|
10ul
|
Buffer 3
|
2.5ul
|
EcoR1
|
0.5ul
|
Pst1
|
0.5ul
|
BSA
|
0.25ul
|
Total
|
25ul
|
3-way
|
260/280
|
ng/ul
|
1T3 (1)
|
1.88
|
56.3
|
1T3 (2)
|
1.91
|
71.5
|
1T3 (3)
|
1.91
|
33.5
|
1T3 (4)
|
1.96
|
20.8
|
1T3 (5)
|
1.87
|
30.1
|
|
|
|
1T3 backbone (1)
|
3.78
|
3.9
|
1T3 backbone (2)
|
1.91
|
5.8
|
1T3 backbone (3)
|
1.77
|
2.0
|
1T3 backbone (4)
|
1.42
|
1.9
|
|
ng/ul
|
1G (1)
|
6.37
|
1G (2)
|
0.04
|
1G (3)
|
0.22
|
1G (4)
|
4.48
|
1G (5)
|
0.04
|
Time (h)
|
LB+IPTG
|
0.4M NaCl +LB +IPTG
|
0.6M NaCl +LB +IPTG
|
0.4M KCl+ LB+ IPTG
|
0.6M KCl + LB +IPTG
|
0
|
0.008
|
0.013
|
0.013
|
0.011
|
0.004
|
0.5
|
0.023
|
0.021
|
0.021
|
0.027
|
0.017
|
1.0
|
0.025
|
0.013
|
0.015
|
0.009
|
0.005
|
1.5
|
0.049
|
0.026
|
0.010
|
0.012
|
0.005
|
2.0
|
0.083
|
0.017
|
0.011
|
0.013
|
0.003
|
2.5
|
0.209
|
0.033
|
0.015
|
0.045
|
0.010
|
3.0
|
0.382
|
0.107
|
0.058
|
0.107
|
0.054
|
3.5
|
0.567
|
0.234
|
0.082
|
0.177
|
0.062
|
4.0
|
0.783
|
0.329
|
0.126
|
0.288
|
0.128
|
4.5
|
0.973
|
0.485
|
0.165
|
0.432
|
0.206
|
5.0
|
1.214
|
0.673
|
0.258
|
0.662
|
0.252
|
5.5
|
1.454
|
0.906
|
0.386
|
0.845
|
0.336
|
6.0
|
1.554
|
1.132
|
0.450
|
1.078
|
0.457
|
![](http://samlo.t0.ru/igem/w701.png)
Buffer 3
|
2.5ul
|
100X BSA
|
0.25ul
|
Eco R1
|
0.5ul
|
Pst 1
|
0.5ul
|
DNA
|
14ul (500ng)
|
H20
|
7.25ul
|
Buffer 3
|
2.5ul
|
100X BSA
|
0.25ul
|
Eco R1
|
0.5ul
|
Pst 1
|
0.5ul
|
DNA
|
4ul (500ng)
|
H20
|
17.25ul
|
|
LB + IPTG
|
LB of 0.4M of NaCl
|
Start culture absorbance (1ml)
|
1.323
|
0.464
|
Time (h)
|
LB+ IPTG
|
LB of 0.4M NaCl + IPTG
|
0
|
0.021
|
0.014
|
0.5
|
0.026
|
0.024
|
1.0
|
0.045
|
0.033
|
1.5
|
0.043
|
0.036
|
2.0
|
0.133
|
0.064
|
2.5
|
0.245
|
0.105
|
3.0
|
0.359
|
0.159
|
3.5
|
0.555
|
0.228
|
4.0
|
0.720
|
0.303
|
4.5
|
0.927
|
0.466
|
5.0
|
0628 (2X)
|
0.618
|
5.5
|
0.846(2X)
|
0.815
|
6.0
|
0.899(2X)
|
0.610(2X)
|
![](http://samlo.t0.ru/igem/w702.png)
- lane 3: 1000-1500bp & 2000-3000bp
- lane 4: 100-200bp
- lane 5: 500-1000bp
Time (h)
|
OD600
|
Cell count (cell/ml)
|
0
|
0.000
|
4 X 108
|
0.5
|
0.003
|
1.4 X 1010
|
1.0
|
0.008
|
8.6 X 107
|
1.5
|
0.022
|
3.3 X 108
|
2.0
|
0.142
|
3 X 108
|
2.5
|
0.203
|
7 X 108
|
3.0
|
0.263
|
/
|
![](http://samlo.t0.ru/igem/stock/image012.png)
|
Ng/ul
|
260/280
|
3-way
|
12.8
|
1.93
|
Cl sensor
|
1.2
|
1.85
|
T71T3
|
13.0
|
1.93
|
HR1C3
|
103.0 (15.9)
|
1.33
|
5A
|
35.3
|
1.87
|
7A
|
24.2
|
1.90
|
![](http://samlo.t0.ru/igem/stock/image013.png)
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
5480
|
5633
|
5428
|
5554
|
3013
|
3267
|
3058
|
3209
|
1249
|
1335
|
1341
|
1267
|
8990
|
9199
|
9483
|
9446
|
5235
|
5222
|
5241
|
4649
|
1545
|
1646
|
1634
|
1630
|
763
|
21493
|
21870
|
23048
|
11411
|
11549
|
11711
|
10767
|
2564
|
2513
|
2549
|
2413
|
25542
|
29517
|
27811
|
29312
|
14561
|
14986
|
14392
|
14646
|
3056
|
3018
|
2848
|
2839
|
30141
|
32470
|
33457
|
27985
|
17182
|
17219
|
14902
|
19335
|
3351
|
3318
|
2974
|
2931
|
37055
|
36110
|
39855
|
35193
|
18985
|
18717
|
18398
|
16975
|
3565
|
3349
|
3216
|
3366
|
34258
|
36477
|
37255
|
35236
|
18320
|
18588
|
18778
|
6182
|
3425
|
3211
|
3424
|
3265
|
36893
|
37123
|
37393
|
35904
|
13574
|
25312
|
19447
|
1334
|
3386
|
3529
|
3191
|
3364
|
A: 1000mM NaCl
|
B: 500mM NaCl
|
C: 100mM NaCl
|
D: 50mM NaCl
|
E: 25mM NaCl
|
F: 12.5mM NaCl
|
G: 6.25mM NaCl
|
H: 3.125mM NaCl
|
|
ng/ul
|
260/280
|
24C (1)
|
44.1
|
1.77
|
24C (2)
|
59.7
|
1.76
|
T71K3 (1)
|
---
|
---
|
T71K3 (2)
|
41.7
|
1.87
|
HR1K3 (1)
|
18.5
|
1.92
|
HR1K3 (2)
|
29.5
|
1.78
|
3A (1)
|
---
|
---
|
3A (2)
|
50.7
|
1.88
|
5A (1)
|
44.4
|
1.85
|
5A (2)
|
---
|
---
|
1G (1)
|
142.9
|
1.86
|
1G (2)
|
52.4
|
1.89
|
7A
|
43.5
|
1.86
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.5ul
|
Spe1
|
0.5ul
|
T71K3 (prepared on 4/8)
|
16.8ul
|
Total
|
20ul
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.5ul
|
Pst 1
|
0.5ul
|
HR1C3 (prepared on 3/8)
|
16.8ul
|
Total
|
20ul
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.5ul
|
Pst 1
|
0.5ul
|
7A (prepared on 3/8)
|
4ul
|
H2O
|
12.8ul
|
Total
|
20ul
|
T4 ligase buffer
|
2ul
|
T4 ligase
|
1ul
|
T7
|
10ul
|
HR
|
4ul
|
Vector (7A)
|
3ul
|
Total
|
20ul
|
Samples prepared on 4/8
|
ng/ul
|
260/280
|
HR1T3 (1)
|
10.3
|
1.98
|
HR1T3 (2)
|
12.2
|
1.91
|
T71T3 (1)
|
10.5
|
1.82
|
T71T3 (2)
|
12.4
|
1.86
|
|
0.4M NaCl LB with light
|
0.4M NaCl LB without light
|
LB with light
|
LB without light
|
OD600 (2X)
|
0.230
|
0.401
|
0.388
|
0.690
|
|
ng/ul
|
260/280
|
3-way ligation
|
11.5
|
1.83
|
HR-T7-1T3 (1)
|
13.9
|
1.86
|
HR-T7-1T3 (2)
|
13.2
|
1.91
|
T71T3 (1), prepared on 6/8
|
29.4
|
1.52
|
T71T3 (2) , prepared on 6/8
|
7.3
|
1.69
|
T71T3 (3) , prepared on 6/8
|
5.4
|
1.74
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.5ul
|
Spe 1
|
0.5ul
|
T71T3 (prepared on 6/8)
|
16.8ul
|
Total
|
20ul
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
XBa 1
|
0.5ul
|
Pst 1
|
0.5ul
|
HR1K3 (prepared on 4/8)
|
16.8ul
|
Total
|
20ul
|
Buffer 2
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.5ul
|
Pst 1
|
0.5ul
|
3A (prepared on 4/8)
|
2ul
|
H2O
|
14.8ul
|
Total
|
20ul
|
T4 ligase buffer
|
2ul
|
T4 ligase
|
1ul
|
T7
|
7ul
|
HR
|
7ul
|
Vector pSB1C3 from 3-way
|
3ul
|
Total
|
20ul
|
|
LB of 0.4M NaClwith light
|
LB of 0.4M NaClwithout light
|
LB with light
|
LB without light
|
OD600 (2X)
|
0.328
|
0.503
|
0.875
|
0.934
|
|
LB of 0.4M NaClwith light
|
LB of 0.4M NaClwithout light
|
LB with light
|
LB without light
|
OD600 (2X)
|
0.357
|
0.526
|
0.390
|
0.627
|
|
ng/ul
|
260/280
|
T71T3 (1)
|
111.0
|
1.84
|
T71T3 (2)
|
109.9
|
1.85
|
T71T3 (3)
|
147.9
|
1.90
|
HR1T3 (1)
|
103.0
|
1.89
|
HR1T3 (2)
|
98.9
|
1.85
|
HR1T3 (3)
|
107.6
|
1.84
|
3A (1)
|
101.2
|
.182
|
3A (2)
|
123.6
|
1.88
|
3A (3)
|
210.0
|
1.87
|
3-way post miniprep (1)
|
3-way post miniprep (2)
|
3-way post miniprep (3)
|
3-way (1)
|
3-way (2)
|
3-way (3)
|
T71T3 (1)
|
T71T3 (2)
|
T71T3 (3)
|
Tube
|
Concentration (ug/ul)
|
260/280
|
3A Post mini prep (1)
|
96.1
|
1.91
|
3A Post mini prep (2)
|
41.6
|
1.96
|
3A Post mini prep (3)
|
100.5
|
1.85
|
3A (1)
|
104.7
|
1.85
|
3A (2)
|
68.8
|
1.87
|
3A (3)
|
74.7
|
1.96
|
T71T3 (1)
|
87.3
|
1.79
|
T71T3 (2)
|
115.8
|
1.85
|
T71T3 (3)
|
135.9
|
1.91
|
labeled as “ transf. 24C, 11/8,ii)
|
LB of 0.4M NaClwith light
|
LB of 0.4M NaClwithout light
|
LB with light
|
LB without light
|
OD600 (2X)
|
0.266
|
0.267
|
0.302
|
0.286
|
|
LB of 0.4M NaClwith light
|
LB of 0.4M NaClwithout light
|
LB with light
|
LB without light
|
OD600 (2X)
|
0.094
|
0.096
|
0.350
|
0.355
|
Volume to bespinned (ml)
|
*All
|
*All
|
4.5
|
4.5
|
|
LB of 0.4M NaClwith light
|
LB of 0.4M NaClwithout light
|
LB with light
|
LB without light
|
OD600 (2X)
|
0.459
|
0.290
|
0.768
|
0.847
|
Volume to bespinned (ml)
|
3.5
|
5.5
|
2
|
1.9
|
set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.857
|
0.724
|
0.733
|
0.316
|
0.660
|
Volume to bespinned down (ml)
|
1.762
|
2.086
|
2.060
|
4.778
|
2.288
|
![](http://samlo.t0.ru/igem/stock/image014.jpg)
set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.862
|
0.242
|
0.751
|
0.797
|
0.252
|
Volume to bespinned down (ml)
|
2.8
|
10
|
3.2
|
3.0
|
9.6
|
![](http://samlo.t0.ru/igem/stock/image015.jpg)
![](http://samlo.t0.ru/igem/stock/image016.jpg)
Buffer
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.4ul
|
Pst1
|
0.4ul
|
T7
|
15ul
|
H20
|
2ul
|
TOTAL
|
20ul
|
Buffer
|
2ul
|
BSA
|
0.2ul
|
EcoR1
|
0.4ul
|
Pst1
|
0.4ul
|
backbone
|
10ul
|
H20
|
7ul
|
TOTAL
|
20ul
|
Lane
|
1
|
2
|
3
|
4
|
5
|
6
|
|
---
|
100bp ladder
|
HR-1
|
HR-1
|
HR-2
|
HR-2
|
T4 ligase buffer
|
2ul
|
T4 ligase
|
1ul
|
T7
|
3ul
|
1C3
|
14ul
|
TOTAL
|
20ul
|
- white colonies: T71C3
- red colonies: 1G1A3, 3A1C3, 5A1K3, 7A1T3
|
ug/ul
|
260/280
|
24C (1)
|
10.5
|
1.72
|
24C (2)
|
7.9
|
1.78
|
24C (3)
|
8.2
|
1.73
|
24C (4)
|
7.9
|
1.59
|
24C (5)
|
11.7
|
1.54
|
24C (6)
|
26.7
|
1.37
|
HR (1)
|
6
|
1.67
|
HR (2)
|
4.9
|
1.80
|
HR (3)
|
8
|
1.07
|
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.303
|
0.350
|
0.637
|
0.689
|
0.269
|
Volume to bespinned down (ml)
|
8
|
6.9
|
3.8
|
3.5
|
9
|
![](http://samlo.t0.ru/igem/stock/image018.png)
![](http://samlo.t0.ru/igem/stock/image020.png)
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.923
|
0.875
|
0.318
|
0.396
|
0.706
|
Volume to bespinned down (ml)
|
2.62
|
2.77
|
7.6
|
6.11
|
3.43
|
![](http://samlo.t0.ru/igem/stock/image022.png)
![](http://samlo.t0.ru/igem/stock/image024.png)
sample
|
Conc. (ng/ul)
|
260/280
|
HR1C3 (1)
|
30.5
|
1.74
|
(2)
|
24.7
|
1.81
|
(3)
|
19.9
|
1.94
|
(4)
|
22.1
|
1.97
|
(5)
|
28.3
|
1.80
|
(6)
|
50.0, 34.8, 21.0, 24.8, 25.3
|
1.94, 1.77, 2.25, 1.97, 1.91
|
(7)
|
42.1, 29.5
|
1.91, 1.92
|
(8)
|
24.6
|
1.78
|
(9)
|
31.4, 25.8
|
1.80, 1.91
|
(10)
|
18.9
|
1.91
|
(11)
|
19.2
|
1.99
|
(12)
|
48.2, 43.1, 45.8
|
1.88, 1.99, 1.92
|
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.667
|
0.148
|
0.107
|
0.178
|
0.181
|
Volume to bespinned down (ml)
|
1.81
|
9.5
|
9.5
|
9.5
|
9.5
|
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.851
|
0.718
|
0.767
|
0.322
|
0.717
|
Volume to bespinned down (ml)
|
3.4
|
4.0
|
3.8
|
9.0
|
4.0
|
![](http://samlo.t0.ru/igem/stock/image026.png)
![](http://samlo.t0.ru/igem/stock/image028.png)
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.878
|
0.326
|
0.447
|
0.262
|
0.625
|
Volume to bespinned down (ml)
|
2.7
|
7.2
|
5.3
|
9.0
|
3.8
|
- HR cut by Xbal1 & Pst1
- T7 cut by EcoR1 & Spe1
- 2 sets, 1 set at 16oC overnight, another set at room temperature, 1hour
- 2 sets, 1 set at 16oC overnight, another set at room temperature, 1hour
- 1set, at 16oC overnight
Lane
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
Sample
|
1bb ladder
|
100bp ladder
|
HR T7
@16 oC
|
HR T7 @22 oC
|
T7 1C3 @16 oC
|
HR 1C3 @22 oC
|
HR 1C3 @16 oC
|
-HR, T7 à correct bands
-HR-T7 à no band
![](http://samlo.t0.ru/igem/stock/image029.jpg)
Sample
|
ng/ul
|
260/280
|
HR
|
12.4
|
1.70
|
T7
|
7.9
|
1.99
|
HR-T7
|
8.0
|
1.57
|
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.946
|
0.264
|
0.302
|
0.359
|
0.631
|
Volume to bespinned down (ml)
|
2.5
|
9.0
|
7.9
|
6.6
|
3.8
|
Set up
|
LB + IPTG without light
|
NaCl + IPTG with light
|
NaCl + IPTG without light
|
KCl + IPTG with light
|
KCl + IPTG without light
|
OD600 (2X)
|
0.301
|
0.588
|
0.454
|
0.269
|
0.468
|
Volume to bespinned down (ml)
|
8
|
4.1
|
5.3
|
9
|
5.2
|
Sample
|
ng/ul
|
260/280
|
HR
|
5.4
|
1.83
|
T7
|
27.1
|
1.73
|
Sample
|
ng/ul
|
260/280
|
HR1C3 @16 oC (1)
|
16.8
|
1.76
|
(2)
|
18.6
|
1.76
|
(3)
|
12.8
|
1.79
|
(4)
|
16.1
|
1.78
|
HR1C3 @22 oC (1)
|
5.7
|
1.96
|
(2)
|
6.9
|
1.69
|
(3)
|
5.2
|
1.77
|
(4)
|
7.5
|
1.64
|
sample
|
ng/ul
|
260/280
|
1
|
46.6
|
1.85
|
2
|
60.5
|
1.81
|
3
|
58.9
|
1.88
|
![](http://samlo.t0.ru/igem/stock/image030.jpg)
![](http://samlo.t0.ru/igem/stock/image032.jpg)
"Creativity is thinking up new things. Innovation is doing new things." - Theodore Levitt
©Copyright CUHK IGEM Team 2011