Team:Groningen/Labwork/13 September 2013

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.