Team:Groningen/Labwork/3 September 2013

Claudio

PCR using as template S5 and primer S16-F along S16-R was performed. This new pair of primers was tested at two diffrent annealing temperature 60°C and 62°C, respectively.
The presence of the expected products was checked using gel electrophoresis (agarose 0.8%).
The PCR worked and the samples were purified.
The PCR product (S5) was digested with XbaI and PstI and purified.

pSB1C3-S1 and pSB1C3-S2 were digested with Spei and PstI.
The samples were checked using gel electrophoresis (agarose 0.8%).

S5 was ligated into pSB1C3-S1 and pSB1C3-S2 (1:1 molar ratio). The ligation reaction was incubated 1h at 37°C and 4°C overnight.

Sander

made an inoculation of s1,2,5,6,7,8,11,12,13 an 14.

Sebas

Performed a PCR on pDR111 for the hyperspank promoter -> checked on gel for right size -> purified the product and
digested it with NheI and EcoRI for 30min at 37degrees.

Digested MunichBB+hyperspank(wrong direction)+GFPdsm/GFP0804/RFP with NheI and EcoRI for 30min at 37degrees

Digested pX(Pxyl) with SpeI and SdaI and column purfied the digested plasmid.

Ligated O/N in beaker with RT water in fridge:

pX(SpeI*SdaI) + S1S13(XbaI*PstI)
MBB-GFPdsm(NheI*EcoRI) + hy_spank(NheI*EcoRI)
MBB-GFP0804(NheI*EcoRI) + hy_spank(NheI*EcoRI)
MBB-Prfp(NheI*EcoRI) + hy_spank(NheI*EcoRI)

Molar ratio of vector:insert was 1:3.