Assembly of endosomal detection operon

Marcin

Task 1:

• Gel-outs of following constructs digested on 17.08.09:
• cro CDS on pKSII plasmid
• BBa_C0040 with BBa_B0032 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:

• Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation

Task 2:

• Ligate cro CDS to pSB1A3 plasmid

Methods:

• Reaction mixture composition:

10 μl insert
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

Task 3:

• Prepare bacterial cultures to isolate following constructs:
• BBa_R0080 on pSB1A3 plasmid
• BBa_C0040 with BBa_B0032 on pSB1A3 plasmid

Task 4:

• Isolate of constructs described in Task 3:

Methods:

• Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 5:

• Digest previously isolated plasmids with C0040+B0032 biobricks:

Methods:

• Reaction mixture composition:

 
35 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
10 μl MQ water

• Digest was performed overnight

Making of the plac-RBS-llo part

Jarek

Tasks:

• Isolation of plasmid DNA from 20 liquid cultures
• Digestion of the plasmid DNA with PstI and EcoRI restriction endonucleases