IGEM:Groningen/Notebook/iGEM 2011/2011/09/13

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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 will be done in one reaction (so insert and vector will be in one tube, so don't use fastAP!)
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

17μl digested purified sample
1μl T4 DNA ligase
2μl T4 DNA ligase buffer

Ligate for 30 to 40 min at room temperature

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