Assembly of endosomal detection operon

Marcin

Task 1:

• Isolation of pSB1A3 plasmids containing BBa_B0032 and BBa_E0022

Methods:

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

Task 2:

• Digest previously isolated plasmids to verify the correctness of the ligation

Methods:

• Reaction mixture composition:

 
1 μl purified plasmid DNA product
0.5 μl XbaI (Fermentas)
0.5 μl PstI (Fermentas)
2 μl Buffer Tango (Fermentas)
16.5 μl MQ water

Comment:

All isolated plasmids had the expected construct.

Task 3:

• Prepare bacterial cultures to isolate following constructs:
• p53 CDS and BBa_B0032 on pSB1A3 plasmid
• cro CDS and BBa_B0032 on pSB1A3 plasmid

Task 4:

• Isolate of following constructs:
• p53 CDS and BBa_B0032 on pSB1A3 plasmid
• cro CDS and BBa_B0032 on pSB1A3 plasmid

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 to verify the correctness of the ligation

Methods:

• Reaction mixture composition:

 
1 μl purified plasmid DNA product
0.5 μl XbaI (Fermentas)
0.5 μl PstI (Fermentas)
2 μl Buffer Tango (Fermentas)
16.5 μl MQ water

Comment:

None of isolated plasmids had the expected constructs. Ligations must be prepared once again.

Task 6:

• Restriction digest of following constructs:
• p53 CDS on pSB1A3 plasmid using XbaI and PstI
• BBa_R0080 on pSB1A3 plasmid using SpeI and PstI
• BBa_E0010 on pSB1A3 plasmid using PstI and XbaI
• BBa_C0040 with RBS on pSB1A3 plasmid

Methods:

• Reaction mixture composition:

 
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas) or 0.5 SpeI (Fermentas)
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Making of the plac-RBS-llo part

Jarek

Tasks:

• *Transformating the competent ''E.coli'' strain TopF' with ligation (acquired from Marek) containing RBS-llo gene cloned into vector with plac promoter
• *Plating the transformated culure on LB+Amp plates