Team:Groningen/14 June 2010

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Week 24, Arend Jan

Control digestion of 4 clones of pNZ8901-bbs (bbs = biobrick sites). The original plasmid does have a XbaI, SpeI, and PstI site (which were replaced in the PCR with these sites in the correct orientation), but no EcoRI site. Therefore the control digestion was done with EcoRI. Also, HindIII was removed in the PCR but not replaced so clones should not be cut with HindIII.

- 10ul plasmid
- 1.5ul buffer EcoRI/R
- 0.5ul EcoRI/HindIII
- 3ul MQ 

Clones 1 and 2. U = undigested plasmid, E = cut with EcoRI, H = cut with HindIII

Clones 3 and 4. Use this imager in the future

EcoRI cuts in all clones except only partially in 4. HindIII does not cut in all clones (positive control would have been nice, new enzyme however). Clones 1, 2, and 3 are good for sure.

David & Peter

Extraction of Streptomyces cellwalls

Harvesting of chaplin coated mycelium of Streptomyces with a razorblade.

Disruption of mycelium.

ExtractionCellwallsGR

Chaplin isolation/purification.

TFA treatment

After extraction the freeze dried cellwalls consist for approx 5% of their dry weight of assembled chaplin proteins. Further purification and monomerisation of the chaplin proteins is achieved by "TFA" treatment of extracted cellwalls. TFA treated chaplins are monomeric and can be rediluted in demiwater or trisbuffer. Note that after redulution the monomeric chaplins will immediately start to reassemble in amyloid fibres. This proces is accelerated by shaking of the liquid.

TFA treatmentGR