Team:Groningen/Labwork/26 July 2013

Mirjam

Made a gel to check the gel purified products of Pdes, CheY and des DOWN. These products look fine. A check of the PCR products of CheY UP, spec and CheY DOWN revealed that the PCR for spec failed again. So a new PCR reaction for spec is made. As well as a gel purification for spec.

Gel purification is done for CheY UP, CheY DOWN and Tet.

New PCR reactions are made for all types of silk we want to create. This time the bp time is increased to 30 sec, to examine if this helps to obtain a higher concentration.

Did a restriction digestion with BamHI for Pdes and CheY. The restriction digestion for Pdes failed. So a new restriction digestion is made. This one worked well. Therefore a ligation reaction is made. A restriction digest is made on this ligation product with EcoRI and PstI. As well as a restriction digestion for BBa_k823823. The restriction digestion for BBa_k823823 went well. But the restriction on the ligation product failed.
A restriction is made for des UP, tet and des DOWN. The restriction for this three products went well. So a ligation is made.

Did a restriction digestion for RFP and eYFP obtained from the biobrick plates. Again no bands appeared.

Claudio

The Bacillus Subtilis plates incubated overnight show colonies.
One colony is picked and transformed with the plasmid LacI promoter in backbone BBa_k823823.
The transformants are plated on:

• LB agar + chloramphenicol
• LB agar + chloramphenicol + IPTG

Other two colonies are picked and transformed, respectively with the construct aimed to knock out the des gene and with the latter along with the modified Munich backbone (see above).
The transformants are plated on:

• LB agar + tetracycline
• LB agar + chloramphenicol + tetracycline

Sander

Did a gel purification of silk without strep tag and ran it on a new gel. 0.8% agarose, 90V, 34min. Unfortunately the samples are lost during purification.