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Team:Groningen/project notebook/13 September 2011
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induction with different arabinose concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 %) <br> | induction with different arabinose concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 %) <br> | ||
measurement over time with plate reader (every 15 min, 40 cycles) | measurement over time with plate reader (every 15 min, 40 cycles) | ||
| + | <br> | ||
| + | '''Joyce''' | ||
| + | <br> | ||
| + | <br> Waiting for the key of the lab. Making presentation for schools and practicing this a little | ||
| + | <br> Checking sequencing results, were not alright for most samples. | ||
| + | <br> | ||
| + | <br> Receive all samples for digestion, ligation and transformation: 28 samples. | ||
| + | <br> PCR product was purified and analysed on gel. Product with annealing temp of 65 tube 2 was alright and used for cloning. | ||
| + | <br> Concentration was low: 20ng/μl | ||
| + | <br> | ||
| + | <br> Digestion: | ||
| + | <br> Digestion will be done in one reaction (so insert and vector will be in one tube, so don't use fastAP!) | ||
| + | <br> Scheme: | ||
| + | <br> 1.5μl pSB1C3 (50 ng) | ||
| + | <br> 6μl plasmid sample | ||
| + | <br> 1μl EcoRI | ||
| + | <br> 1μl PstI | ||
| + | <br> 2μl FD buffer | ||
| + | <br> 8.5μl MQ water | ||
| + | <br> | ||
| + | <br> incubate samples for 1.5h at 37 degrees | ||
| + | <br> | ||
| + | <br> Clean up of the digested samples with the High Pure PCR Purification Kit | ||
| + | <br> NOTE: elute in 17 μl MQ water! So it can be used for ligation immediately | ||
| + | <br> | ||
| + | <br> Ligation: | ||
| + | <br> 17μl digested purified sample | ||
| + | <br> 1μl T4 DNA ligase | ||
| + | <br> 2μl T4 DNA ligase buffer | ||
| + | <br> | ||
| + | <br> Ligate for 30 to 40 min at room temperature | ||
| + | <br> | ||
| + | <br> Transformation | ||
| + | <br> Add to 40μl competent cells 10μl ligation mixture | ||
| + | <br> Incubate for 30 min on ice | ||
| + | <br> Heat shock: 45s at 42 degrees | ||
| + | <br> Incubate cells on ice for 2 min | ||
| + | <br> Add 1ml LB medium+ 25mM glucose | ||
| + | <br> Incubate cells for 1h/1.5h at 37 degrees | ||
| + | <br> Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates | ||
| + | <br> Put the plates in the stove at 37 degrees overnight | ||
| + | <br> Finished at 8 o'clock | ||
| + | <br> | ||
| + | |||
{{FooterGroningen2011}} | {{FooterGroningen2011}} | ||
Latest revision as of 15:24, 20 September 2011
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Olesja
measurement of construct: PBad_RBS_GFP_DT (colony 4 & 6 + control cells)
induction with different arabinose concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 %)
measurement over time with plate reader (every 15 min, 40 cycles)
Joyce
Waiting for the key of the lab. Making presentation for schools and practicing this a little
Checking sequencing results, were not alright for most samples.
Receive all samples for digestion, ligation and transformation: 28 samples.
PCR product was purified and analysed on gel. Product with annealing temp of 65 tube 2 was alright and used for cloning.
Concentration was low: 20ng/μl
Digestion:
Digestion will be done in one reaction (so insert and vector will be in one tube, so don't use fastAP!)
Scheme:
1.5μl pSB1C3 (50 ng)
6μl plasmid sample
1μl EcoRI
1μl PstI
2μl FD buffer
8.5μl MQ water
incubate samples for 1.5h at 37 degrees
Clean up of the digested samples with the High Pure PCR Purification Kit
NOTE: elute in 17 μl MQ water! So it can be used for ligation immediately
Ligation:
17μl digested purified sample
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
Ligate for 30 to 40 min at room temperature
Transformation
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
Finished at 8 o'clock
