IGEM:Groningen/Notebook/iGEM 2011/2011/09/13: Difference between revisions
Joyce Mulder (talk | contribs) (Autocreate 2011/09/13 Entry for IGEM:Groningen/Notebook/iGEM_2011) |
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== | ==13-9-11== | ||
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<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> | |||
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Revision as of 01:20, 15 September 2011
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13-9-11
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