Cloning of cro gene into pSB+ vector

Kama

• chemocompetent E. coli TOP10 were incubated on ice for 15 minutes
• 20μl of ligation mixture was added
• bacteria were incubated with DNA on ice for 30 minutes
• heat shock was conducted (1min30sec 42°C)
• bacteria were incubated on ice for 3 minutes
• After the heat shock 780μl of SOB medium was added
• Mixture with bacteria was incubated for 1 hour in 37°C
• 100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG (diluted 10* and without dilution)

Assembly of endosomal detection operone/Assembly of fusion proteins

Marcin

Task 1:

• Digest plasmids isolated in 13.09.2009 to reveal the identity of the constructs

Methods:

• Reaction mixture composition:

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

• Digestion was carry out 2 hours

Results: There is no evidence for occurence of signal peptide CDS. The situation of [http://partsregistry.org/Part:BBa_K177037BBa_K177037] biobricks is unclear since lack of expected restriction pattern

Comment:

Because of short length of signal peptide it is possible that I am incapable of finding this fragment on the gel. I think the sequencing of some samples may reveal the identity of the both signal peptide and [http://partsregistry.org/Part:BBa_K177037BBa_K177037]

Task 2:

Comment:

Due to some problem which our ampicilline I decided to use vector with resistance to kanamycine instead of previously use [http://partsregistry.org/Part:pSB1A3pSB1A3]

• Prepare the backbone plasmid [http://partsregistry.org/Part:pSB1K3pSB1K3] to ligation with COX mitochondrial signal.

• Reaction mixture composition:

20 μl purified plasmid DNA product
1 μl PstI (Fermentas)
1 μl SpeI (Fermentas)
5 μl Buffer Tango (Fermentas)
23 μl MQ water

• Digestion was carry out 4 hours

Result:

The digestion was severe incomplete. Most of DNA was uncut. It is obligated to carry out the reaction longer.