Team:Groningen/21 June 2010

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Week 25, Arend Jan

Control restriction of pNZ8901-bbs with biobrick site enzymes. All were also cut with XhoI to check the orientation of the sites (in case the sites in the original plasmid were not removed).

- 10ul plasmid
- 1.5ul buffer G (XbaI, SpeI) or R (PstI)
- 0.5ul biobrick enzyme (XbaI, SpeI, or PstI)
- 0.5ul XhoI
- 2.5ul MQ 

Clones 1 and 2. + = cut with XhoI, P+ = cut with PstI and XhoI, S+ = cut with SpeI and XhoI, X+ = cut with XbaI and XhoI

Clones 3 and 4.

Because of the added XhoI a ~270bp fragment should be present. This is the case in all restrictions. All PstI restrictions give an unexpected 3 bands. The plasmid is ~800bp larger than the clone manager file but this was not considered a problem. It is likely that there is a PstI site in the unknown sequence. This site will have to be deleted before we can use this plasmid in multi-biobrick cloning steps.

As an extra control a restriction was done with BamHI which was used to linearize the plasmid after inserting the biobrick sites. Together with XhoI this should give a ~300bp band.

- 5ul plasmid
- 1.5ul buffer G
- 0.5ul BamHI
- 0.5ul XhoI
- 7.5ul MQ 

3* = clone 3 only cut with BamHI

300bp band is present. The biobrick sites seem to be correctly inserted. The construct will be sequenced at a later stage.

The plasmid contains an unknown sequence with an ‘illegal’ PstI site which we will need to delete. To approximate the position of the unknown sequence a restriction was performed with PstI and SalI + PstI.

- 10ul plasmid
- 1.5 buffer O
- 0.5/1ul enzymes (PstI or PstI + SalI(NEB))
- 3/2.5ul MQ 

PstI site in unknown sequence is ~1100bp away form the PstI site in the biobrick suffix. The unknown sequence could still be either downstream of the SalI site or downstream of repC.