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 iGEM promotor oligo's
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To Do
- DONE Prepare competent TOP10 Cells
- DONE Prepare fresh CaCl2 (0.1 M) (0.3319 g in 30 mL demi)
- DONE Look for filters for sterilization (0.2 μm Whattman filter used to sterilize CaCl2, CaCl2 was put on ice)
- DONE Look for canister for liquid nitrogen
- DONE 60 mL (6·10 mL in 50 mL greiner tubes were inoculated with 100 μL ON culture TOP10 cells)
- DONE Isolate pSB1AC3-H & pSB3K3-H plasmids
- DONE Transform ligations
- DONE Use each batch of 20 mL and give 2 batches only positive control
Competent cells
- 5 mL ON culture is used to inoculate 6·10 mL of LB, 100 μL per 10 mL.
- Cultures are grown @ 37 °C untill an OD600 of 0.2 ~ 0.3 is reached.
- Cultures are spinned down @ 4000 rpm, 4 °C
- Supernatant is removed and pellet is resuspended in 10 mL chilled 0.1 M CaCl2
- Suspension is incubated on ice for 10 min.
- Suspensions are spinned down @ 4000 rpm, 4 °C
- Supernatant is removed and pellet is resuspended in 1770 μL chilled 0.1 M CaCl2 and supplemented with 230 μL 87% glycerol prior to making aliquots.
- Cells are divided in 50 μL aliquotes
- Cells are snapfrozen in liquid nitrogen and stored @ -80 °C
OD600
| Culture
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t = 1.25 h
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t = 1.75 h
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Cell batch
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Quality
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| 1
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0.119
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0.307
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| 2
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0.113
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0.304
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A
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Good
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| 3
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0.108
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0.290
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| 4
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0.113
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0.301
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B
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Good
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| 5
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0.140
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0.315
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| 6
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0.120
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0.321
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C
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Good
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Plasmid isolation
Plasmids pSB3K3-H and pSB1AC3-H were isolated using (NucleoSpin® Plasmid, Machery nagel])
Concentration was determined using nanodrop
'
| Vector
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ng/μg
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260/280
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260/230
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| pSB1AC3-H
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116.0
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1.80
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1.29
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| pSB3K3-H
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23.4
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1.36
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0.34
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Control competence
- All batches (A, B & CC) were transformed with 1 μL pSB1AC3 isolated today
Oligo's
- Ligations were transformed in batch B of competent cells, 5 μL ligation mix in 50 μL competent TOP10 cells.
- 200 μL of LB was added for 1 h of recovery @ 37 °C
- Suspensions were plated out on TY Amp100 plates
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iGEM promotor oligo's
