Team:Macquarie Australia/Protocols/GibsonAssembly
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- | <center>Before entering the lab, the assigned Gibson Assembly team for the day organised agar plate requirements, concentration calculations and equipment requirements. </center> | + | <center>Before entering the lab, the assigned Gibson Assembly team for the day organised agar plate requirements, concentration calculations and equipment requirements. All gBlocks had arrived and thus Gibson Assembly was performed on all fragments.</center> |
<center>Each tube was labelled with antibiotic (C = Chloramphenicol, K = Kanamycin, A = Ampicillin) resistance contained within the allocated plasmid. Tubes labeled 1 & 2 represent Heme Oxygenase fragment mixtures, tubes labeled 3 represent Deinococcus fragment mixtures & tubes labeled 4 & 5 represent Agrobacterium fragment mixtures. Take note that #1C and #4C contain T7 promoter fragments whereas the other BioBricks are T7 'promoter-less'. Deinococcus BioBrick does not contain any T7 promoters as described [https://2012.igem.org/Team:Macquarie_Australia/Protocols/ArrivalofGBlocks here.] | <center>Each tube was labelled with antibiotic (C = Chloramphenicol, K = Kanamycin, A = Ampicillin) resistance contained within the allocated plasmid. Tubes labeled 1 & 2 represent Heme Oxygenase fragment mixtures, tubes labeled 3 represent Deinococcus fragment mixtures & tubes labeled 4 & 5 represent Agrobacterium fragment mixtures. Take note that #1C and #4C contain T7 promoter fragments whereas the other BioBricks are T7 'promoter-less'. Deinococcus BioBrick does not contain any T7 promoters as described [https://2012.igem.org/Team:Macquarie_Australia/Protocols/ArrivalofGBlocks here.] |
Revision as of 10:52, 5 September 2012
Gibson Assembly Protocol
#1C | #2K | #2A | #3K | #3A | #4C | #5K | #5A | |
---|---|---|---|---|---|---|---|---|
1. Addition of 20ng Gene Block Fragments in appropriate tube | 2µl Hemo_T7A | 2µl Hemo_A | 2µl Hemo_A | 2µl Deino_A | 2µl Deino_A | 2µl Agro_T7A | 2µl Agro_A | 2µl Agro_A |
2µl Hemo_B | 2µl Hemo_B | 2µl Hemo_B | 2µl Deino_B | 2µl Deino_B | 2µl Agro_B | 2µl Agro_B | 2µl Agro_B
| |
nil | nil | nil | 2µl Deino_C | 2µl Deino_C | 2µl Agro_C | 2µl Agro_C | 2µl Agro_C
| |
nil | nil | nil | 2µl Deino_D | 2µl Deino_D | 2µl Agro_D | 2µl Agro_D | 2µl Agro_D
| |
nil | nil | nil | 2µl Deino_E | 2µl Deino_E | 2µl Agro_E | 2µl Agro_E | 2µl Agro_E | |
2. Addition of 0.05 pmol of vector | 2.7µl PSB-1C3 | 2.9µl PSB-1K3 | 2.8µl PSB-1A3 | 2.9 µl PSB-1K3 | 2.8 µl PSB-1A3 | 2.7µl PSB-1C3 | 2.9µl PSB-1K3 | 2.8µl PSB-1A3 |
3. Addition of Gibson Master Mix (µl) | 10 | 10 | 10 | 12.9 | 12.8 | 12.7 | 12.9 | 12.8 |
4. Addition of deionised H2O | 3.3µl | 3.1µl | 3.2µl | nil | nil | nil | nil | nil |
5. After addition of all components incubation at 50°C for 60 min followed.