Team:Groningen/Labwork/9 September 2013

Claudio

S13 and S14 were digested with XbaI and PstI and purified.
pSB1C3-S1-S5 and pSB1C3-S2-S5 were digested with SpeI and PstI.
pSB1C3-S1-S5 and pSB1C3-S2-S5 were ligated to S13 and S14, respectively.
The ligation product was transformed in E. Coli DH5α and plated on LB + Cm.

S3 and S9 were digested with BamHI and PstI.
S16 was digested with EcoRI and BamHI.
S16 was ligated to both S3 and S9.
The ligation reaction was run on gel 0.8% to check the presence of product.
The gel didn't show any significant band around the expected height. However the product was ligated subsequently into the backbone (pSB1C3) and the ligation was incubated over night at 4°C.

Mirjam

Prepared the silk samples for sequencing.
Did a colony PCR twice to get the ΔDes and ΔCheY out of their initial strain. This was not successful.

Sebas

Did a PCR on plasmids from the green and red colonies (GFPdsm 2&3, GFP0840 3&4, RFP 2&3) with Primer FW that binds in
the promoter and a primer RV that binds outside the promoter.
Products were formed but not migrated properly through the gel, so no conclusions could be drawn.

Sent GFPdsm-3, GFP0840-3, RFP-4 for sequencing with hyspank_FW and Hyspank_RV primers. Transformed those plasmids also to B. subtilis.

EZ-seq codes:
152DZAB037 GFP0840 3 F
152DZAB038 GFP0840 3 R
152DZAB039 GFPdsm 3 F
152DZAB040 GFPdsm 3 R
152DZAB041 RFP 4 F
152DZAB042 RFP 4 R