Team:Groningen/Labwork/30 July 2013

Mirjam

Started gel extraction for the restricted backbone+promoter, RFP and eYFP. RFP and eYFP are dissolved in 30µl elution buffer, to get a higher concentration
Run a 0.8% agarose gel to examine the restriction. Gel revealed only a very small band for RFP.
Did a nanodrop on the samples to examine their concentration. Concentration of the backbone is 0.8 ng/µl, RFP 6.4 ng/µl and eYFP 2.4 ng/µl.
Did a new restriction digestion for BBa_k823823 + LacI with EcoRI and PstI, using 500 and 250ng.

Chaline & Mirjam

Did a restriction digestion for eYFP with EcoRI and PstI using 200ng.
Run the samples of restricted eYFP and the backbone on gel for gel extraction. But the concentration of the eYFP looks again very low. For all samples the band is extracted from gel. Concentration is measured and again the concentration seems way to low, for what we expected. The samples are run on gel to determine if the number of bp is the expected one. No bands are seen on gel. But trusted the concentrations and continued with a ligation reaction.
Transformation to E. coli with half of the ligation of the backbone with eYFP and the ligation of the backbone with RFP. Also made a new transformation to obtain more RFP and eYFP.
Did an examination on the restriction products of CheY UP and CheY DOWN. Gel examination revealed that the products are still present. The concentration is measured using nanodrop.
Streaked out B. subtilis strain 168 cells on a plate of LB agar. To perform eventually a transformation to B. subtilis

Claudio

The transformation of BBa_E0840 and BBa_E0240 worked because cells grew in LB + chloramphenicol. The inoculation of mKate2 worked.
Plasmids are isolated from the inoculations.
BBa_K823023 + lacI, BBa_E0840, BBa_E0240 and mKate2 are digested with XbaI and PstI.
The digestion of BBa_K823023 + lacI didn't work so we stopped.

E. Coli containing BBa_K823023 + lacI was inoculated in LB + Ampicillin in order to isolate the plasmid the day after.

Sander

Did a miniprep for BBa_E0240, BBa_E0840 and mKate2, DNA concentations to low.
inoculated the Des knockout strains in LB agare with Cm 5 ug/ml and Kn 5ug /ml. cells are to grow overnight at 37 C.

Inne

Inoculated tyhe OIB055 strains which are cheY null on LB + cm plates and in LB medium. cells are to grow overnight at 37 C