Team:NYMU-Taipei/Project/Lab Note

From 2009.igem.org

Contents

Experiment of oscillator biobrick parts

1 BBa_C0051 cI repressor from E. coli phage lambda (+LVA)
2 BBa_K116602 CII coding region from λ phage
3 BBa_C0040 tetracycline repressor from transposon Tn10 (+LVA)
4 BBa_C0074 penI repressor from Bacillus licheniformis (+LVA)
5 BBa_C0072 mnt repressor (strong) from Salmonella phage P22 (+LVA)
6 BBa_C0073 mnt repressor (weak) from Salmonella phage P22 (+LVA)
7 BBa_E0040 green fluorescent protein
t BBa_B0015 Terminator
A BBa_R0040 TetR repressible promoter
P BBa_R0074 Promoter (PenI regulated)
L BBa_R0010 promoter (lacI regulated)
M BBa_R0073 Promoter (Mnt regulated)
C BBa_R0051 promoter (lambda cI regulated)
pCI BBa_R1051 Promoter, Standard (lambda cI regulated)
p22 BBa_R0053 Promoter (p22 cII regulated)
pLuxR BBa_R0062 Promoter (luxR & HSL regulated -- lux pR)
pLasR BBa_R0079 Promoter (LasR & PAI regulated)
E BBa_K116603 pRE promoter from λ phage



Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: BBa_R0051 pCI 287bp (49+238) 238bp
2: BBa_C0051 CI lam 988bp (750+238) 238bp
3: BBa_C0040 TetR 838bp (660+238) 238bp
4: BBa_B0034 RBS 250bp (12+238) 238bp
5: BBa_j13002 pTetR 302bp (74+238) 238bp
6: BBa_B0015 Term 445bp (129+316) 316bp
7: BBa_K116602 CII 532bp (294+238) 238bp
8: BBa_K116603 pRE 286bp (48+238) 238bp
9: Positive Control BBa_E0240 1114bp (876+238) 238bp
10: Negative Control 0bp
11: marker 100bp


Experiment of oscillator biobrick parts (2)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: BBa_R0040 pTetR 292bp (54+238) 238bp
2: BBa_E0840 1116bp (878+238) 238bp
3: CII<Term(from jesse) 669bp (294+8+129+238) 532bp (294+238)
4: Positive Control BBa_E0240 1114bp (876+238) 238bp
5: Negative Control Contamination ~300bp
6: - - -
7-8: BBa_B0015 Term (gel extraction) 155bp (129+26)
9: marker 100bp


Experiment of oscillator biobrick parts (3)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: CII<Term 669bp (294+8+129+238) 532bp (294+238)
2: CI lam<Term 1125bp (750+8+129+238) 988bp (750+238)
3: TetR<Term 1035bp (660+8+129+238) 898bp (660+238)
4: BBa_R0051 pCI 287bp (49+238) 238bp
5: BBa_j13002 pTetR+RBS 312bp (74+238) 238bp
6: Positive Control BBa_E0240 1114bp (876+238) 238bp
7: Negative Control 0bp Contamination ~300&700bp,
8: - - -
9-10: BBa_E0840 (Gel extraction) 878bp
11: marker 100bp

4: Inconclusive.

Experiment of oscillator biobrick parts (4)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: BBa_R0074 pPenI 315bp (77+238) 238bp
2-6: BBa_C0074 PenI 739bp (423+316) 316bp
7-11: BBa_R0073 pMnt 383bp (67+316) 316bp
12-16: BBa_C0072 Mnt (Strong) 604bp (288+316) 316bp
17-21: BBa_C0073 Mnt (Weak) 604bp (288+316) 316bp
22: Positive Control BBa_E0240 1114bp (876+238) 238bp
23: Negative Control 0bp Contamination
24: marker 100bp

The result for lanes 7-11 look weird, but it could be because of the gel. A rerun will be done.


Experiment of oscillator biobrick parts (5)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1-2: TetR<Term (Gel extraction) 821bp (660+8+129+24) 684bp (660+24)
4-5: CII<Term (Gel extraction) 455bp (294+8+129+24) 318bp (294+24)
7-9: CI<Term (Gel extraction) 911bp (750+8+129+24) 774bp (750+24)
10: marker 100bp


Experiment of oscillator biobrick parts (6)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: BBa_R0074 pPenI 315bp (77+238) 238bp
2: BBa_C0074 PenI 739bp (423+316) 316bp
3: BBa_R0073 pMnt 383bp (67+316) 316bp
4: BBa_C0072 Mnt (Strong) 604bp (288+316) 316bp
5: BBa_C0073 Mnt (Weak) 604bp (288+316) 316bp
6: Positive Control BBa_E0240 1114bp (876+238) 238bp
7: Negative Control 0bp Contamination
8: marker 100bp


Experiment of oscillator biobrick parts (7)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: RBS + CII + Term 687bp 250bp
2: RBS + CI lam + Term 1143bp 250bp
3: RBS + TetR + Term 1053bp 250bp
4: pCI(plate from 090729) 287bp 238bp
5: E0840 604bp (288+316) 316bp
6: pCI(plasmid from 090730) 287bp 238bp
7: Positive Control BBa_E0240 1114bp (876+238) 238bp
8: Negative Control 0bp Contamination~300bp
9: marker 1kb+100bp


Experiment of oscillator biobrick parts (8)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: BBa_I13504 r7t 1113bp 250bp
2: BBa_I13522 Ar7t 1176bp 292bp
3: BBa_I13401 7t 1095bp 958bp
4: BBa_R0051 pCI 287bp 238bp
5: pTetR<E0240 1176bp 292bp
6: PenI<Term 876bp 739bp
7: Mnt (Strong)<Term 741bp 604bp
8: Mnt (Weak)<Term 741bp 604bp
9: Positive Control BBa_E0240 1114bp (876+238) 238bp
10: Negative Control 0bp Contamination~300bp
11: marker 100bp


Experiment of oscillator biobrick parts (9) Gel Extraction


2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: B0034+CII+Term r2t 475bp 38bp
2: B0034+CII+Term r2t 475bp 38bp
3: B0034+CII+Term r2t 475bp 38bp
4: B0034+CI lam+Term r1t 931bp 38bp
5: B0034+CI lam+Term r1t 931bp 38bp
6: B0034+CI lam+Term r1t 931bp 38bp
7: B0034+Tet R+Term r3t 841bp 38bp
8: B0034+Tet R+Term r3t 841bp 38bp
9: B0034+Tet R+Term r3t 841bp 38bp
10: marker 100bp


Experiment of oscillator biobrick parts (10) (Gel Extraction)


 2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: BBa_I13504 r7t 901bp 0bp
2: BBa_I13401 7t 881bp 0bp
3: PenI+Term 584bp 447bp
4: Mnt (Strong)+Term 5t 449bp 312bp
5: Mnt (Weak)+Term 6t 449bp 312bp
6: TetR+Term 3t 821bp 684bp
7: marker 1kb+100bp


Experiment of oscillator biobrick parts (11)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: marker 1kb
2: C 287bp 238bp
3: M 383bp 316bp
4: 3t 1035bp 898bp
5: 4t 876bp 739bp
6: 5t 741bp 604bp
7: 6t 741bp 604bp
8: Positive Control E0240 1114bp 238bp
9: Negative Control 0bp Contamination~300
10: bp bp
11: bp bp
12: r<1t 1143bp 250bp
13: r<2t 687bp 250bp
14: r<7t 1113bp 250bp
15: 3<t 1035bp 898bp
16: 5<t 741bp 604bp
17: Positive Control E0240 1114bp 238bp
18: Negative Control 0bp Contamination~
19: marker 1kb
20: marker 100bp


Experiment of oscillator biobrick parts (12)


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: Lr7t 1321bp 438bp
2: Lr7t 1321bp 438bp
3: Positive Control E0240 1114bp 238bp
4: r1t 1143bp 250bp
5: r1t 1143bp 250bp
6: r1t 1143bp 250bp
7: r1t 1143bp 250bp
8: r1t 1143bp 250bp
9: r1t 1143bp 250bp
10: r1t 1143bp 250bp
11: r1t 1143bp 250bp
12: r2t 687bp 250bp
13: r2t 687bp 250bp
14: r2t 687bp 250bp
15: r2t 687bp 250bp
16: r2t 687bp 250bp
17: r2t 687bp 250bp
18: r2t 687bp 250bp
19: r2t 687bp 250bp
20: r2t 687bp 250bp
21: r2t 687bp 250bp
22: r2t 687bp 250bp
23: Negative Control 0bp
24: marker 1kb+100bp


Experiment of oscillator biobrick parts (13) 2009/08/13


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 90V, 38min

Descr Win length Fail length
0: marker 1kb+100bp
1: 3t 1035bp 898bp
2: 3t 1035bp 898bp
3: 3t 1035bp 898bp
4: 3t 1035bp 898bp
5: 3t 1035bp 898bp
6: 4t 876bp 739bp
7: 4t 876bp 739bp
8: 4t 876bp 739bp
9: 4t 876bp 739bp
10: 4t 876bp 739bp
11: 5t 741bp 604bp
12: 5t 741bp 604bp
13: 5t 741bp 604bp
14: 5t 741bp 604bp
15: 5t 741bp 604bp
16: 6t 741bp 604bp
17: 6t 741bp 604bp
18: 6t 741bp 604bp
19: 6t 741bp 604bp
20: 6t 741bp 604bp
21: 1t 1125bp 988bp
22: Positive Control E0240 1114bp 238bp
23: Negative Control 0bp
24: marker 1kb+100bp


Experiment of oscillator biobrick parts (14) (Gel Extraction) 2009/08/13


 2% agarose, 90V, 36min

Descr Win length Fail length
0: marker 1kb+100bp
1: r1t 929bp
2: r1t 929bp
3: marker 1kb+100bp
4: r2t 473bp
5: r2t 473bp


Experiment of oscillator biobrick parts (15) 2009/08/15


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: Ar7t 1176bp 292bp
2: Ar7t 1176bp 292bp
3: Ar7t 1176bp 292bp
4: Ar2t 749bp 292bp
5: Pr7t 1199bp 315bp
6: Lr7t 1322bp 438bp
7: Lr7t 1322bp 438bp
8: Lr7t 1322bp 438bp
9: Lr7t 1322bp 438bp
10: Cr7t 1171bp 287bp
11: Cr7t 1171bp 287bp
12: Cr7t 1171bp 287bp
13: Cr7t 1171bp 287bp
14: Er7t 1170bp 286bp
15: Er7t 1170bp 286bp
16: Er7t 1170bp 286bp
17: Er7t 1170bp 286bp
18: Er7t 1170bp 286bp
19: Positive Control E0240 1114bp 238bp
23: Negative Control 0bp
24: marker 1kb+100bp


Experiment of oscillator biobrick parts (16) (Gel Extraction) 2009/08/15


 2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: 3t 821bp 684bp
2: 3t 821bp 684bp
3: 3t 821bp 684bp
4: 4t 584bp 447bp
5: 4t 584bp 447bp
6: 4t 584bp 447bp
7: marker 1kb+100bp


Experiment of oscillator biobrick parts (17) 2009/08/16


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: pH sensor 512bp
2: pH sensor 512bp
3: pH sensor 512bp
4: pH sensor 512bp
5: r3t 1053bp 250bp
6: r3t 1053bp 250bp
7: r3t 1053bp 250bp
8: r3t 1053bp 250bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp
11: marker 1kb+100bp


Experiment of oscillator biobrick parts (18) (digestion) 2009/08/17


 2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 1kb
1: marker 100bp
2: Cr7t(XN) 311,2701bp 253bp,2701bp
3: Er7t(NP) 36,274,2701bp 253bp,2701bp
4: pH Sensor Plasmid #1 Amplify 1-2 (XP) 296,2057bp 1180,2057bp
5: pH Sensor Plasmid #1 Amplify 3-4 (XP) 296,2057bp 1180,2057bp
6: pH Sensor Plasmid #1 Amplify 1-2 (XN) 2353bp 536,2701bp
6: pH Sensor Plasmid #1 Amplify 3-4 (XN) 2353bp 536,2701bp
7: marker 1kb+100bp

Comments

  • Colony's of lanes 4-7 came from the same plate. Because this part was left over from last year, we didn't know if this plasmid contained K116001 or K116002. This gel's digestion results say K116002.


Experiment of oscillator biobrick parts (19) (Gel Extraction) 2009/08/17


 2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 100bp
1: marker 1kb
2: Er7t 958bp 74bp
3: Er7t 958bp 74bp
4: Er7t 958bp 74bp
5: Cr7t 959bp 75bp
6: Cr7t 959bp 75bp
7: Cr7t 959bp 75bp
8: marker 1kb
9: marker 100bp


Experiment of oscillator biobrick parts (20) 2009/08/18


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: AR7t 1175bp 292bp
2: AR7t 1175bp 292bp
3: PR7t 1198bp 315bp
4: PR7t 1198bp 315bp
5: CR7t 1170bp 287bp
6: CR7t 1170bp 287bp
7: pMnt 383bp 316bp
8: CI lam 988bp 238bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp contamination~700bp
11: marker 1kb+100bp


Experiment of oscillator biobrick parts (21) (Gel Extraction) 2009/08/18


 2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: r3t(XP) 841bp 38bp
2: r3t(XP) 841bp 38bp
3: r3t(XP) 841bp 38bp
4: term(XP) 155bp 445bp
5: term(XP) 155bp 445bp
6: term(XP) 155bp 445bp
7: marker 1kb+100bp


Experiment of oscillator biobrick parts (22) 2009/08/19


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
pfu 0.5μl
ddH20 39.5μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 60s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: nhaA 274bp 0bp
2: Positive Control Term 445bp 316bp
3: Negative Control 0bp
4: marker 1kb+100bp

PCR of nhaA using this protocol worked.


Experiment of oscillator biobrick parts (23) 2009/08/19


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: 4<t 876bp 739bp
2: 4<t 876bp 739bp
3: 4<t 876bp 739bp
4: 4<t 876bp 739bp
5: 5<t 741bp 604bp
6: 5<t 741bp 604bp
7: 5<t 741bp 604bp
8: 5<t 741bp 604bp
9: 6<t 741bp 604bp
10: 6<t 741bp 604bp
11: 6<t 741bp 604bp
12: 6<t 741bp 604bp
13: M<R7t 1267bp 383bp
14: M<R7t 1267bp 383bp
15: M<r7t 1267bp 383bp
16: M<r7t 1267bp 383bp
17: M 383bp 316bp
18: P 315bp 238bp
19: 1 988bp 238bp
20: 5 604bp 316bp
21: 6 604bp 316bp
22: Positive Control E0240 1114bp 238bp
23: Negative Control 0bp contamination~300,1100bp
24: marker 1kb+100bp


Experiment of oscillator biobrick parts (24) 2009/08/20


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 27min

Descr Win length Fail length
0: marker 1kb+100bp
1: nhaA -->(l) 512bp 238bp
2: A<r2t 749bp 292bp
3: E<r3t 1109bp 286bp
4: E<r3t 1109bp 286bp
5: E<r3t 1109bp 286bp
6: 4t 876bp 739bp
7: 5t 741bp 604bp
8: 6t 741bp 604bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp
11: marker 1kb+100bp

Comments:

  • Next time, for the failed ligations, revert back to using old enzymes and redo. Maybe it's something to do with not being familiar enough with the new brands.
  • Ligating digested PCR product nhaA(XP) onto an empty pSB1A2 plasmid (obtained from extracting it from digesting E0240(XP)) worked.


Experiment of oscillator biobrick parts (25) 2009/08/21


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: E<r3t-->(l) 1109bp 286bp
2: E<r3t 1109bp 286bp
3: E<r3t 1109bp 286bp
4: E<r3t 1109bp 286bp
5: E<r3t 1109bp 286bp
6: E<r3t 1109bp 286bp
7: E<r3t 1109bp 286bp
8: E<r3t 1109bp 286bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp contamination~700,1100,1400
11: marker 1kb+100bp


Comments: liquid incubation of number 1


Experiment of oscillator biobrick parts (26) 2009/08/22


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: P<r3t 1138bp 315bp
2: P<r3t 1138bp 315bp
3: P<r3t 1138bp 315bp
4: P<r3t 1138bp 315bp
5: 4<t 876bp 739bp
6: 4<t 876bp 739bp
7: 4<t 876bp 739bp
8: 4<t-->(4) 876bp 739bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp contamination~700
11: marker 1kb+100bp


Comments:

  • 4t in lane 5 seems strange. It is not correct,but not fail either. Lane 7 and 8 are correct. They could do liquid incubation.
  • There are no colony on the yesterday's plate Ar2t, so there is no need to do colony PCR.
  • Ligation&Transformation: Ar7t、Pr7t、Lr7t(33.3uL competent cell each)


Experiment of oscillator biobrick parts (27) 2009/08/23 colony PCR


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: A<r7t-->(1) 1176bp 292bp
2: A<r7t 1176bp 292bp
3: P<r7t-->(2) 1199bp 315bp
4: P<r7t 1199bp 315bp
5: L<r7t-->(3) 1322bp 438bp
6: L<r7t 1322bp 438bp
7: P<r3t 1138bp 315bp
8: P<r3t 1138bp 315bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp
11: marker 1kb+100bp


Comments:

  • all the colonies that are transformed yesterday are all correct. Pr3t is from the checking plate 090821.
  • haven't done the liquid incubation yet, should I do the liquid?
  • Lr7t is green enough to be identified. Ar7t and Pr7t are not sure enough for me to identify.
  • r7t is BBa_I13504 from Biobrick, not the ligation from r<7t.
  • Today's transformation :
    • Ar<2t
    • Ar<7t
  • Ar is BBa_J13002 from Biobrick.


Experiment of oscillator biobrick parts (28) 2009/08/23 testing the reheating gel


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: r1t 1176bp 292bp
2: CI lam 988bp 238bp
3: pMnt 1199bp 315bp
4: unknown bp bp
5: unknown bp bp
6: unknown bp bp
7: marker 1kb+100bp


Comments:

  • This gel is made by many pieces of gel and be reheat once. The result seems that this gel could be use to check DNA length, not suitable for gel extraction.
  • CI lam is from the old PCR tube. The result is not that reliable since it is not fresh enough. But the result is strange because it is not success or failure either.
  • pMnt seems to be right since that the right marker is upper than th left one, so the length is not sure.

Experiment of oscillator biobrick parts (29) 2009/08/26


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 29min

Descr Win length Fail length
0: marker 1kb+100bp
1: Pr3t 1138bp 315bp
2: Pr3t 1138bp 315bp
3: Pr3t 1138bp 315bp
4: Pr3t 1138bp 315bp
5: Pr3t 1138bp 315bp
6: Pr3t 1138bp 315bp
7: Pr3t 1138bp 315bp
8: Pr3t 1138bp 315bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp Contamination~1400bp
11: marker 1kb+100bp


Comments:

  • It is not that correct since those colony have not 100% correct.
  • Pr3t in lane 2 seems to be correct in the upper band, while it also has the wrong length band.

Experiment of oscillator biobrick parts (29) 2009/08/27


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 29min

Descr Win length Fail length
0: marker 1kb+100bp
1: Pr3t 1138bp 315bp
2: Pr3t 1138bp 315bp
3: Pr3t 1138bp 315bp
4: Pr3t 1138bp 315bp
5: Pr3t 1138bp 315bp
6: nhaA+ E0240 1396bp 512bp
7: nhaA+ E0240 1396bp 512bp
8: nhaA+ E0240 1396bp 512bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp Contamination~1400bp
11: marker 1kb+100bp


Comment:

  • all of those bands are fail. It means that nhaA+E0240 didn't ligate well.
  • DO the ligation again?


Experiment of oscillator biobrick parts (30) 2009/08/28


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 29min

Descr Win length Fail length
0: marker 1kb+100bp
1: Pr3t 1138bp 315bp
2: Pr3t 1138bp 315bp
3: Pr3t 1138bp 315bp
4: Pr3t 1138bp 315bp
5: Pr3t 1138bp 315bp
6: Pr3t 1138bp 315bp
7: Ar2t 749bp 292bp
8: Ar2t 749bp 292bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp
11: marker 1kb+100bp


Comments:

  • It is great progress that we have finally done the ligation of the Ar2t. Doing the liquid this time, including correct one and fail one.
  • Pr3t is correct. Doing the liquid culture tomorrow from the checking plate 090828.


Experiment of oscillator biobrick parts (31) 2009/08/31


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: Ar2t<Er7t 1689bp 749bp
2: Ar2t<Er7t 1689bp 749bp
3: Ar2t<Er7t 1689bp 749bp
4: Ar2t<Er7t 1689bp 749bp
5: Ar2t<Er7t 1689bp 749bp
6: Ar2t<Er7t 1689bp 749bp
7: Ar2t<Er7t 1689bp 749bp
8: Ar2t<Er7t 1689bp 749bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp Contamination~700bp
11: marker 1kb+100bp


Comments:

  • Because the ligation is fail, I restarted from the ligation again. I changed the protocol, which I used 5.5uL inserted DNA instead of using 4uL inserted DNA.
  • Since there are still have colony on the plate, we will run colony PCR again with the ligation I did today(L<r7t and Ar2t<Er7t.


Experiment of oscillator biobrick parts (32) Gel Extraction 2009/08/31


2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: 4t 584bp 447bp
2: 4t 584bp 447bp
3: 4t 584bp 447bp
4: 4t 584bp 447bp
5: r2t 475bp 38bp
6: r2t 475bp 38bp
7: r2t 475bp 38bp
8: r2t 475bp 38bp
9: marker 1kb+100bp


Comments:

  • The failure of 4t is due to the digestion, so we have to digest it again.
  • r2t is fine, so we have extract them from the gel.


Experiment of oscillator biobrick parts (33) 2009/09/01


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 29in

Descr Win length Fail length
0: marker 1kb+100bp
1: Ar2t<Er7t(090830 ligation) 1689bp 749bp
2: Ar2t<Er7t(090830 ligation) 1689bp 749bp
3: Ar2t<Er7t(090830 ligation) 1689bp 749bp
4: Ar2t<Er7t(090830 ligation) 1689bp 749bp
5: Ar2t<Er7t(090830 ligation) 1689bp 749bp
6: Ar2t<Er7t(090830 ligation) 1689bp 749bp
7: Ar2t<Er7t(090830 ligation) 1689bp 749bp
8: L<r2t 895bp 438bp
9: L<r2t 895bp 438bp
10: L<r2t 895bp 438bp
11: L<r2t 895bp 438bp
12: L<r2t 895bp 438bp
13: L<r2t 895bp 438bp
14: L<r2t 895bp 438bp
15: Ar2t<Er7t(090831 ligation) 1689bp 749bp
16: Ar2t<Er7t(090831 ligation) 1689bp 749bp
17: Ar2t<Er7t(090831 ligation) 1689bp 749bp
18: Ar2t<Er7t(090831 ligation) 1689bp 749bp
19: Ar2t<Er7t(090831 ligation) 1689bp 749bp
20: Ar2t<Er7t(090831 ligation) 1689bp 749bp
21: Ar2t<Er7t(090831 ligation) 1689bp 749bp
22: Positive Control E0240 1114bp 238bp
23: Negative Control 0bp contamination~250,1400bp
24: marker 1kb+100bp


Comments:

  • Liquid incubation should be done by the next day.
  • I have do the liquid of the 9 and 19. Only 19 is correct. I will do the Digestion and Ligation tomorrow.



Experiment of oscillator biobrick parts (34) Gel Extraction 2009/09/02


2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: Ar2tEr7t 1476bp 537bp
2: Ar2tEr7t 1476bp 537bp
3: Ar2tEr7t 1476bp 537bp
4: Ar2t 537bp 80bp
5: Ar2t 537bp 80bp
6: Ar2t 537bp 80bp
7: marker 1kb+100bp


Comments:

  • The failure of Ar2t is strange, maybe it is that I put too much DNA in the digestion.
  • Ar2tEr7t is not digested well, next time we should not put too much DNA.
  • concentration measurement before the digestion



Experiment of oscillator biobrick parts (35) 2009/09/03


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 140s
3. 72oC: 300s
2% agarose, 100V, 29min

Descr Win length Fail length
0: marker 1kb+100bp
1: Er3t<Ar2tEr7t 2567bp 1109bp
2: Er3t<Ar2tEr7t 2567bp 1109bp
3: Er3t<Ar2tEr7t 2567bp 1109bp
4: Er3t<Ar2tEr7t 2567bp 1109bp
5: Er3t<Ar2tEr7t 2567bp 1109bp
6: Er3t<Ar2tEr7t 2567bp 1109bp
7: Er3t<Ar2tEr7t 2567bp 1109bp
8: Er3t<Ar2tEr7t 2567bp 1109bp
9: Positive Control E0240 1113bp 250bp
10: Negative Control 0bp Contamination~290bp,1300bp,1400bp
11: marker 1kb+100bp


Comments:

  • There is a lot of contamination in the negative control. We speculated that it might be due to buffer since that eyelight used the old taq and buffer.
  • The length seems to be right, but it is still need to be measured by GFP machine to check if it could be oscillate.


Experiment of oscillator biobrick parts (36) Gel Extraction 2009/09/03


2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 1kb+100bp
1: Ar2t(8-2) 537bp 80bp
2: Ar2t(8-2) 537bp 80bp
3: Ar2t(8-2) 537bp 80bp
4: Er3t 897bp 74bp
5: Er3t 897bp 74bp
6: Er3t 897bp 74bp
7: -
8: 4t 584bp 447bp
9: 4t 584bp 447bp
10: 4t 584bp 447bp
11: marker 1kb+100bp


Comments:

  • Ar2t still fails. Er3t is the one that we are trying to ligate with Ar2t.


Experiment of oscillator biobrick parts (37) 2009/09/04


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 110s
3. 72oC: 300s
2% agarose, 100V, 29in

Descr Win length Fail length
0: marker 1kb+100bp
1: Lr2t<Er7t 1834bp 895bp
2: Lr2t<Er7t 1834bp 895bp
3: Lr2t<Er7t 1834bp 895bp
4: Lr2t<Er7t 1834bp 895bp
5: Lr2t<Er7t 1834bp 895bp
6: P<r2t 772bp 315bp
7: P<r2t 772bp 315bp
8: P<r2t 772bp 315bp
9: P<r2t 772bp 315bp
10: P<r2t 772bp 315bp
11: Ar2t<Er3t 1628bp 749bp
12: Ar2t<Er3t 1628bp 749bp
13: Ar2t<Er3t 1628bp 749bp
14: Ar2t<Er3t 1628bp 749bp
15: Ar2t<Er3t 1628bp 749bp
16: r<4t 816bp 250bp
17: r<4t 816bp 250bp
18: r<4t 816bp 250bp
19: r<4t 816bp 250bp
20: r<4t 816bp 250bp
21: r<4t 816bp 250bp
22: Positive Control E0240 1114bp 238bp
23: Negative Control 0bp
24: marker 1kb+100bp


Comments

  • There is no contamination since I use the old buffer and enzyme. In addition, I change to use new dNTP and I speculated the contamination might be due to new buffer.
  • Need to make liquid incubation by using checking plate


Experiment of oscillator biobrick parts (38) Gel Extraction 2009/09/09


2% agarose, 100V, 28min

Descr Win length Fail length
0: marker 100bp
1: marker 1kb
2: r4t 604bp 38bp
3: r4t 604bp 38bp
4: r4t 604bp 38bp
5: -
6: Ar2tEr7t 707bp  ?bp
7: Er3tAr2tEr7t 707bp  ?bp
8: marker 1kb
9: marker 100bp


Comments:

  • r4t is correct though it is not digested well enough.



Experiment of oscillator biobrick parts (39) 2009/09/11


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 110s
3. 72oC: 300s
2% agarose, 100V, 28in

Descr Win length Fail length
0: marker 1kb+100bp
1: A<r4t 878bp 292bp
2: E<r4t 872bp 286bp
3: E<r4t 872bp 286bp
4: E<r4t 872bp 286bp
5: E<r4t 872bp 286bp
6: E<r4t 872bp 286bp
7: E<r4t 872bp 286bp
8: Pr2t<Er7t 1712bp 772bp
9: Pr2t<Er7t 1712bp 772bp
10: Pr2t<Er7t 1712bp 772bp
11: Pr2t<Er7t 1712bp 772bp
12: Pr2t<Er7t 1712bp 772bp
13: Pr2t<Er7t 1712bp 772bp
14: Positive Control E0240 1114bp 238bp
15: Negative Control 0bp
16: marker 1kb+100bp


Comments

  • It is awful that the plate of Ar4t only have one colony on it. Maybe it is due to the bad digestion of pTet(A). Should the experiment be started from the digestion of pTet or ligation?
  • Pr2t<Er7t could do the colony PCR again to check if there are another correct colony.



Experiment of oscillator biobrick parts (40) 2009/09/15


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 100s
3. 72oC: 300s
2% agarose, 100V, 29in

Descr Win length Fail length
0: marker 1kb+100bp
1: Pr2t<Er7t 1712bp 772bp
2: Pr2t<Er7t 1712bp 772bp
3: Pr2t<Er7t 1712bp 772bp
4: Pr2t<Er7t 1712bp 772bp
5: Pr2t<Er7t 1712bp 772bp
6: A<r4t 878bp 292bp
7: A<r4t 878bp 292bp
8: A<r4t 878bp 292bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp contamination~700bp
11: marker 1kb+100bp


Comments:

  • Redo the colony PCR again and use more colony to check if there is a correct colony.
  • Lane 5 is strange, maybe it is the P<Er7t because the r2t hasn't been ligated correctly.
  • Ar4t keeps fail, however there are some small colony we can pick up.



Experiment of oscillator biobrick parts (41) 2009/09/17


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 110s
72oC: 100s
3. 72oC: 300s
2% agarose, 100V, 29in

Descr Win length Fail length
0: marker 1kb+100bp
1: A<r4t 878bp 292bp
2: A<r4t 878bp 292bp
3: A<r4t 878bp 292bp
4: A<r4t 878bp 292bp
5: A<r4t 878bp 292bp
6: A<r4t 878bp 292bp
7: A<r4t 878bp 292bp
8: A<r4t 878bp 292bp
9: A<r4t 878bp 292bp
10: A<r4t 878bp 292bp
11: A<r4t 878bp 292bp
12: Pr2t<Er7t 1712bp 772bp
13: Pr2t<Er7t 1712bp 772bp
14: Pr2t<Er7t 1712bp 772bp
15: Pr2t<Er7t 1712bp 772bp
16: Pr2t<Er7t 1712bp 772bp
17: Pr2t<Er7t 1712bp 772bp
18: Pr2t<Er7t 1712bp 772bp
19: Pr2t<Er7t 1712bp 772bp
20: Pr2t<Er7t 1712bp 772bp
21: Pr2t<Er7t 1712bp 772bp
22: Positive Control E0240 1114bp 238bp
23: Negative Control 0bp contamination~300bp,700bp
24: marker 1kb+100bp


Comments:


Experiment of oscillator biobrick parts (42) 2009/09/18


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 40s
3. 72oC: 300s
2% agarose, 100V, 28in

Descr Win length Fail length
0: marker 1kb+100bp
1: pLuxR 293bp 238bp
2: pLuxR 293bp 238bp
3: pCI 287bp 238bp
4: pCI 287bp 238bp
5: p22 292bp 238bp
6: p22 292bp 238bp
7: pLasR 395bp 238bp
8: pLasR 395bp 238bp
9: Positive Control Term 445bp 316bp
10: Negative Control 0bp contamination~700bp
11: marker 1kb+100bp


Experiment of oscillator biobrick parts (43) 2009/09/22


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 70s
3. 72oC: 300s
2% agarose, 100V, 28in

Descr Win length Fail length
0: marker 100bp
1: p22+r7t 1175bp 292bp
2: p22+r7t 1175bp 292bp
3: p22+r7t 1175bp 292bp
4: p22+r7t 1175bp 292bp
5: pLuxR+r7t 1176bp 293bp
6: pLuxR+r7t 1176bp 293bp
7: pLuxR+r7t 1176bp 293bp
8: pLuxR+r7t 1176bp 293bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp
11: marker 100bp


Experiment of oscillator biobrick parts (44) 2009/09/24


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28in

Descr Win length Fail length
0: marker 100bp
1: pLuxR+r7t 1176bp 293bp
2: pLuxR+r7t 1176bp 293bp
3: pLuxR+r7t 1176bp 293bp
4: pCI+r7t 1170bp 287bp
5: pLasR+r7t 1278bp 395bp
6: pLasR+r7t 1278bp 395bp
7: pLasR+r7t 1278bp 395bp
8: pLasR+r7t 1278bp 395bp
9: Positive Control E0240 1114bp 238bp
10: Negative Control 0bp contamination~1100bp
11: marker 100bp


Comments:

  • It is strange that the colony of pCI+r7t is glowing obviously, but the length seems to be wrong.


Experiment of oscillator biobrick parts (45) (Gel Extraction) 2009/09/28


 2% agarose, 100V, 30min

Descr Win length Fail length
0: marker 1kb+100bp
1: Pr2tEr7t-18 1500bp 560bp
2: Pr2tEr7t-18 1500bp 560bp
3: Pr2tEr7t-18 1500bp 560bp
4: Pr2tEr7t-19 1500bp 560bp
5: Pr2tEr7t-19 1500bp 560bp
6: Pr2tEr7t-19 1500bp 560bp
7: unknown 841bp 38bp
8: unknown 841bp 38bp
9: unknown 841bp 38bp
10: unknown 841bp 38bp
11: marker 1kb+100bp



Experiment of oscillator biobrick parts (46) 2009/10/17


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 28in

Descr Win length Fail length
0: marker 100bp
1: Er4t<Pr2t 1414bp 872bp
2: Er4t<Pr2t 1414bp 872bp
3: Er4t<Pr2t 1414bp 872bp
4: Er4t<Pr2t 1414bp 872bp
5: Er4t<Pr2t 1414bp 872bp
6: Er4t<Pr2t 1414bp 872bp
7: Er4t<Pr2t 1414bp 872bp
8: Er4t<Pr2t 1414bp 872bp
9: Positive Control Lr7t 1321bp 438bp
10: Negative Control 0bp contamination~1100bp
11: marker 100bp


Experiment of oscillator biobrick parts (47) 2009/10/18


Mix: 50μl
template 1μl
forward primer VF2 1μl
reverse primer VR 1μl
dNTP 2μl
10x buffer 5μl
taq 0.25μl
ddH20 39.75 μl
Protocol:
1. 94oC: 60s
2.
30 cycles 		94oC: 15s
55oC: 20s
72oC: 80s
3. 72oC: 300s
2% agarose, 100V, 30in

Descr Win length Fail length
0: marker 100bp
1: marker 1kb
2: Ar4t<Pr3t 1786bp 878bp
3: Ar4t<Pr3t 1786bp 878bp
4: Ar4t<Pr3t 1786bp 878bp
5: Er4t<Pr2t 1414bp 872bp
6: Er4t<Pr2t 1414bp 872bp
7: Er4t<Pr2t 1414bp 872bp
8: Er4t<Pr2t 1414bp 872bp
9: Er4t<Pr2t 1414bp 872bp
10: Er4t<Pr2t 1414bp 872bp
11: Er4t<Pr2t 1414bp 872bp
12: Er4t<Pr2t 1414bp 872bp
13: Er4t<Pr2t 1414bp 872bp
14: Positive Control Ar7t 1175bp 292bp
15: Negative Control 0bp contamination~1100bp
16: Negative Control 0bp contamination~1100bp
17: marker 1kb
18: marker 100bp
Retrieved from "http://2009.igem.org/Team:NYMU-Taipei/Project/Lab_Note"