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Team:DTU-Denmark/Notebook/30 August 2013
From 2013.igem.org
30 August 2013
Contents |
lab 208
Main purpose
- User reaction
- PCR to amplify HAO and cycAX USER fragments
Who was in the lab
Procedure
USER reaction
Redid USER reaction for ligating our inserts into the Biobrick backbone. There was no cycAX fragment left.
HAO in pSB1C3 AMO in pSB1C3
PCR to amplify HAO and cycAX USER fragments
Made 6 reactions for each, using either the extraction PCR fragment or the glycerol stock as template
| label | cyc gly D | cyc gly % | cyc gly 1uL D | cyc ext D | cyc ext % | cyc ext 1uL D |
|---|---|---|---|---|---|---|
| template | glycerol stock | glycerol stock | glycerol stock | extraction PCR fragment | extraction PCR fragment | extraction PCR fragment |
| DMSO | 1,5 | - | 1,5 | 1,5 | - | 1,5 |
| MgCl2 | - | - | 1 | - | - | 1 |
| MQ | 1 | 2,5 | - | 1 | 2,5 | - |
| label | HAO gly D | HAO gly % | HAO gly 1uL D | HAO ext D | HAO ext % | HAO ext 1uL D |
|---|---|---|---|---|---|---|
| template | glycerol stock | glycerol stock | glycerol stock | extraction PCR fragment | extraction PCR fragment | extraction PCR fragment |
| DMSO | 1,5 | - | 1,5 | 1,5 | - | 1,5 |
| MgCl2 | - | - | 1 | - | - | 1 |
| MQ | 1 | 2,5 | - | 1 | 2,5 | - |
Program:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:20 | 36 |
| annealing temperature | 1:00 | 36 |
| 72C | extension time | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
HAO - 59C, 3:00
cycAX - 55C, 1:30
Results
Gel
- 1 kb ladder
- psB1C3 for Nir, 2% DMSO
- psB1C3 for Nir, 2% DMSO, 1 uL 50mM MgCl2
- psB1C3 for Nir, 4% DMSO
- psB1C3 for Nir, 4% DMSO, 1 uL 50mM MgCl2
- psB1C3 for Nir, 1M Betaine
- purification of psB1C3 USER fragment to make Biobricks (escaped from well -> Ethanol?)
- purification of HAO fragment (escaped from well -> Ethanol?)
- purification of AMO fragment (escaped from well -> Ethanol?)
- purification of psB1C3 USER fragment to make Biobricks (used more loading buffer)
- purification of HAO fragment (used more loading buffer)
- purification of AMO fragment (used more loading buffer)
- purification of pZA21::ara(tight)
- purification of pZA21::ara(tight) with endings for Nir
- 1 kb ladder
Conclusion
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