Line 226: | Line 226: | ||
Dilution pEN: 1/10 (2 uL pEN + 18 uL Elution Buffer EB) final concentration 3.32 ug/mL<br> | Dilution pEN: 1/10 (2 uL pEN + 18 uL Elution Buffer EB) final concentration 3.32 ug/mL<br> | ||
<br> | <br> | ||
− | Component 20 μl Reaction Final Concentration | + | Component 20 μl Reaction Final Concentration<br> |
− | Nuclease free water 3.9 μl | + | Nuclease free water 3.9 μl <br> |
− | 2X Phusion Master Mix 10 1x | + | 2X Phusion Master Mix 10 1x<br> |
− | 5 μM Forward Primer 2 0.5 μM | + | 5 μM Forward Primer 2 0.5 μM<br> |
− | 5 μM Reverse Primer 2 0.5 μM | + | 5 μM Reverse Primer 2 0.5 μM<br> |
− | Template DNA (3.3 ug/mL or ng/uL) 1.5 5 ng | + | Template DNA (3.3 ug/mL or ng/uL) 1.5 5 ng<br> |
− | DMSO (optional) 0.6 3% | + | DMSO (optional) 0.6 3%<br> |
<br> | <br> | ||
Program Gent: IGEM1601<br> | Program Gent: IGEM1601<br> | ||
− | Step Temperature Time | + | <br> |
− | Denaturation 98°C 30s | + | Step Temperature Time<br> |
− | Annealing and elongation 98°C 30s | + | Denaturation 98°C 30s<br> |
− | 61.6°C_ Gradient 2 °C 15s | + | Annealing and elongation 98°C 30s<br> |
− | 72°C 15s | + | 61.6°C_ Gradient 2 °C 15s<br> |
− | Final elongation 72°C 5 min | + | 72°C 15s<br> |
− | Hold 4°C - | + | Final elongation 72°C 5 min<br> |
+ | Hold 4°C - <br> | ||
<br> | <br> | ||
<u>BSU9716 liquid culture prep</u> <br> | <u>BSU9716 liquid culture prep</u> <br> | ||
<br> | <br> | ||
− | |||
− | |||
− | |||
− | |||
Line 276: | Line 273: | ||
− | M 1 2 3 4 5 6 7 | + | M 1 2 3 4 5 6 7 <b>ADD PICTURE</b><br> |
<b>Note:</b> Have same amount of DNA in all well and this was supposed to be a gradient PCR. <br> Therefore, it looks like all temperature are working well. Next PCR simple instead of gradient then if don’t work will do gradient<br> | <b>Note:</b> Have same amount of DNA in all well and this was supposed to be a gradient PCR. <br> Therefore, it looks like all temperature are working well. Next PCR simple instead of gradient then if don’t work will do gradient<br> | ||
Run PCR for fragment A and B<br> | Run PCR for fragment A and B<br> | ||
− | Component 20 μl Reaction Final Concentration | + | Component 20 μl Reaction Final Concentration<br> |
− | Nuclease free water 4.4 μl | + | Nuclease free water 4.4 μl <br> |
− | 2X Phusion Master Mix 10 1x | + | 2X Phusion Master Mix 10 1x<br> |
− | 5 μM Forward Primer 2 0.5 μM | + | 5 μM Forward Primer 2 0.5 μM<br> |
− | 5 μM Reverse Primer 2 0.5 μM | + | 5 μM Reverse Primer 2 0.5 μM<br> |
− | Template DNA (initial concentration?) 1 Don’t know how much DNA | + | Template DNA (initial concentration?) 1 Don’t know how much DNA<br> |
− | DMSO (optional) 0.6 3% | + | DMSO (optional) 0.6 3%<br> |
+ | <br> | ||
Program A IGEM1602<br> | Program A IGEM1602<br> | ||
− | Step Temperature Time | + | <br> |
− | Denaturation 98°C 30s | + | Step Temperature Time<br> |
− | Annealing and elongation 98°C 30s | + | Denaturation 98°C 30s<br> |
− | 66.2°C 15s | + | Annealing and elongation 98°C 30s<br> |
− | 72°C 15s | + | 66.2°C 15s<br> |
− | Final elongation 72°C 5 min | + | 72°C 15s<br> |
− | Hold 4°C - | + | Final elongation 72°C 5 min<br> |
+ | Hold 4°C -<br> | ||
+ | <br> | ||
Program B: IGEM1603 65.9 (remember to change the name it’s IGEM1604 on the thermos cycler) <br> | Program B: IGEM1603 65.9 (remember to change the name it’s IGEM1604 on the thermos cycler) <br> | ||
− | Step Temperature Time | + | <br> |
− | Denaturation 98°C 30s | + | Step Temperature Time<br> |
− | Annealing and elongation 98°C 30s | + | Denaturation 98°C 30s<br> |
− | 65.9 °C 15s | + | Annealing and elongation 98°C 30s<br> |
− | 72°C 15s | + | 65.9 °C 15s<br> |
− | Final elongation 72°C 5 min | + | 72°C 15s<br> |
− | Hold 4°C - | + | Final elongation 72°C 5 min<br> |
+ | Hold 4°C -<br> | ||
+ | <br> | ||
<b>Note:</b> issue with PCR of B; when looked at the TC to check evolution there was an error and we assumed the beginning of the PCR was done so we just did the final elongation step. <br> | <b>Note:</b> issue with PCR of B; when looked at the TC to check evolution there was an error and we assumed the beginning of the PCR was done so we just did the final elongation step. <br> | ||
Run gel for fragment A and B (second half of the 1.2% Gel of the morning) <br> | Run gel for fragment A and B (second half of the 1.2% Gel of the morning) <br> | ||
Line 320: | Line 322: | ||
− | + | M A B M A B <b>ADD PICTURE</b><br> | |
− | + | ||
− | M A B M A B | + | |
Line 331: | Line 331: | ||
<u>PCR Fragments A, B and Gent</u><br> | <u>PCR Fragments A, B and Gent</u><br> | ||
Used diluted samples and oligos done for previous PCR<br> | Used diluted samples and oligos done for previous PCR<br> | ||
− | Component 60 μl Reaction Final Concentration | + | Component 60 μl Reaction Final Concentration<br> |
− | Nuclease free water 13.2 μl | + | Nuclease free water 13.2 μl <br> |
− | 2X Phusion Master Mix 30 1x | + | 2X Phusion Master Mix 30 1x<br> |
− | 5 μM Forward Primer 6 0.5 μM | + | 5 μM Forward Primer 6 0.5 μM<br> |
− | 5 μM Reverse Primer 6 0.5 μM | + | 5 μM Reverse Primer 6 0.5 μM<br> |
− | Template DNA (initial concentration?) 3 Don’t know how much DNA | + | Template DNA (initial concentration?) 3 Don’t know how much DNA<br> |
− | DMSO (optional) 1.8 3% | + | DMSO (optional) 1.8 3%<br> |
PCR programs: A IGEM1602, B IGEM1603 and Gent IGEM1601 without gradient<br> | PCR programs: A IGEM1602, B IGEM1603 and Gent IGEM1601 without gradient<br> | ||
<br> | <br> | ||
Line 345: | Line 345: | ||
<br> | <br> | ||
− | M A B G | + | M A B G<b>ADD PICTURE</b><br> |
Line 353: | Line 353: | ||
<br> | <br> | ||
− | |||
<br> | <br> | ||
<u>Recombinant PCR AG</u><br> | <u>Recombinant PCR AG</u><br> | ||
Used diluted samples and oligos done for previous PCR<br> | Used diluted samples and oligos done for previous PCR<br> | ||
− | Component 60 μl Reaction 1 60 μl Reaction 2 Final Concentration | + | Component 60 μl Reaction 1 60 μl Reaction 2 Final Concentration<br> |
− | Nuclease free water - - | + | Nuclease free water - - <br> |
− | 2X Phusion Master Mix 30 30 1x | + | 2X Phusion Master Mix 30 30 1x<br> |
− | 5 μM Forward Primer DF1A 6 6 0.5 μM | + | 5 μM Forward Primer DF1A 6 6 0.5 μM<br> |
− | 5 μM Reverse Primer RR 6 6 0.5 μM | + | 5 μM Reverse Primer RR 6 6 0.5 μM<br> |
− | Template DNA A 6.6 8.2 (should have been 9.2) ? | + | Template DNA A 6.6 8.2 (should have been 9.2) ?<br> |
− | Template DNA Gent 6.6 6 ? | + | Template DNA Gent 6.6 6 ?<br> |
− | DMSO (optional) 1.8 1.8 3% | + | DMSO (optional) 1.8 1.8 3%<br> |
+ | <br> | ||
PCR programs: A-Gent IGEM1601 without gradient (Ta= 61.6) <br> | PCR programs: A-Gent IGEM1601 without gradient (Ta= 61.6) <br> | ||
<br> | <br> | ||
<u>Recombinant PCR AGB</u><br> | <u>Recombinant PCR AGB</u><br> | ||
Used purified AG 1 and 2 <br> | Used purified AG 1 and 2 <br> | ||
− | + | <br> | |
− | Component 60 μl Reaction 1 60 μl Reaction 2 Final Concentration | + | Component 60 μl Reaction 1 60 μl Reaction 2 Final Concentration<br> |
− | Nuclease free water 1.2 1.2 - | + | Nuclease free water 1.2 1.2 -<br> |
− | 2X Phusion Master Mix 30 30 1x | + | 2X Phusion Master Mix 30 30 1x<br> |
− | 5 μM Forward Primer DF1A 6 6 0.5 μM | + | 5 μM Forward Primer DF1A 6 6 0.5 μM<br> |
− | 5 μM Reverse Primer RR 6 6 0.5 μM | + | 5 μM Reverse Primer RR 6 6 0.5 μM<br> |
− | Template DNA A 6 6 ? | + | Template DNA A 6 6 ?<br> |
− | Template DNA Gent 6 6 ? | + | Template DNA Gent 6 6 ?<br> |
− | DMSO (optional) 1.8 1.8 3% | + | DMSO (optional) 1.8 1.8 3%<br> |
PCR programs: A-Gent IGEM1604 (Ta= 61.8) <br> | PCR programs: A-Gent IGEM1604 (Ta= 61.8) <br> | ||
<br> | <br> |
Revision as of 14:01, 18 October 2016