Team:Macquarie Australia/Protocols/GibsonAssembly
From 2012.igem.org
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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. </center> | ||
- | <center>Each tube was labelled with antibiotic (C = | + | <center>Each tube was labelled with antibiotic (C = Chloramphenicol, K = Kanamycin, A = Ampicillin). 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.] |
Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. </center> | Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. </center> | ||
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+ | After addition of all components incubation at 50°C for 60 min followed. | ||
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[[File:IMG 6181.JPG|325 px|thumb|center|Performing Gibson Assembly]] | [[File:IMG 6181.JPG|325 px|thumb|center|Performing Gibson Assembly]] |
Revision as of 10:48, 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 |
After addition of all components incubation at 50°C for 60 min followed.