Assembly of endosomal detection operon

Marcin

Task 1:

• Gel-outs of following constructs digested on 17.08.09:
• p53 CDS on pSB1A3 plasmid
• BBa_R0080 on pSB1A3 plasmid
• BBa_E0010 on pSB1A3 plasmid
• BBa_C0040 with RBS on pSB1A3 plasmid

Methods:

• All samples were inactivated via heating in 80 °C for 20 minutes
• All gel-outs were performed using the EurX gel-out kit according to the manual

Results:

• All gel-outs (except C0040 with RBS) were successful. One isolation failed because of very low DNA concentration

Task 2:

• Digest pKSII plasmid with cro CDS

Methods:

• Reaction mixture composition:

 
45 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
0.5 μl SpeI (Fermentas)
5 μl Buffer Tango (Fermentas)

Task 3:

• Ligate p53 CDS to pSB1A3 plasmid with BBa_B0032

Methods:

• Reaction mixture composition:

10 μl p53 CDS
7 μl vector
2.3 μl Buffer Tango (Fermentas)
3 μl dNTPs mixture (EurX)
1 μl T4 ligase (Fermentas)

• Ligation was performed for about 9 hours and it was subsequently thermally inactivated

Making of plac-RBS-llo part

Jarek

Tasks:

• Preparation of 20 liquid cultures, from colonies that grew from ligation, in 3 ml of LB+Amp nad incubating them in 37C degree

Cloning switch 1 regulatory parts [ K177012-PcI.RBS.LacI, K177033-PcI.RBS.LacI.PcI.RBS.RFP.terminator, K177011-PLacI.RBS.cI.terminator, K177038-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator ] into two compatible low copy number plasmids of different antibiotic resistance

Ania

Tasks: