Team:Groningen/Labwork/13 September 2013
From 2013.igem.org
Claudio
The plates pSB1C3-S16-3-5 and pSB1C3-S16-9-11 showed around thousands of colonies.Colony PCR was performed (NEB Mater Mix) on 4 colonies per plate using VF2 and VR primers (annealing temperature 64°C).
Colony C from pSB1C3-S16-3-5 and colony D from pSB1C3-S16-9-11 were positive candidate and were inoculated in LB + Cm.
Plates (work previously done by Mirjam) containing several combination of signal sequences together with S3 or S9 showed colonies.
Colony PCR was performed (NEB Mater Mix) on 2 colonies per plate using VF2 and VR primers (annealing temperature 64°C).
The samples were checked on agarose gel 0.8% (Inne).
Colony 1A, 2A, 5A, 7A-B and 8A showed to be positive candidates and were inoculated in LB + Cm.
pSB-S1-S5-S5 and pSB-S2-S5-S5 were digested with SpeI and PstI and the product was purified.
S13 and S14 were ligated into pSB1C3-S1-S5 and pSB1C3-S2-S5, respectively.
The ligation products were transformed into E. coli DH5α and plated on LB + Cm.
Inne
Ran 2 gels with colony pcr samples.Gel 1:
Ladder | s16-3-5 A | s16-3-5 B | s16-3-5 C | s16-3-5 D | s16-9-11 A | s16-9-11 B | s16-9-11 C | s16-9-11 D | Positive control |
Gel 2
Ladder | 1 A | 1 B | empty | empty | 2 A | 2 B | 3 A | 3 B | 4 A | 4 B |
Ladder | 5 A | 5 B | 6 A | 6 B | 7 A | 7 B | 8 A | 8 B | empty | empty |
Sebas
Sequenced RFP strain form 11-09 was not the right one. So did a colony pcr on 16 other colonies still stored in the fridge.Picked a colony stroke it in a PCR tube, than on a plate. 4/16 colonies had the right insert.