Team:Warsaw/Calendar-Main/14 September 2009

Cloning of cro gene into pSB+ vector Kama  chemocompetent E. coli TOP10 were incubated on ice for 15 minutes 20&mu;l of ligation mixture was added bacteria were incubated with DNA on ice for 30 minutes heat shock was conducted (1min30sec 42&deg;C) bacteria were incubated on ice for 3 minutes After the heat shock 780&mu;l of SOB medium was added Mixture with bacteria was incubated for 1 hour in 37&deg;C 100&mu;l of mixture was plated on medium containing ampicillin, X-Gal and IPTG (diluted 10* and without dilution)</li> </ul>

Assembly of endosomal detection operone/Assembly of fusion proteins Marcin Task 1: Methods: 2 μl purified plasmid DNA product 1 μl PstI (Fermentas) 1 μl XbaI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
 * Digest plasmids isolated in 13.09.2009 to reveal the identity of the constructs
 * Reaction mixture composition:
 * Digestion was carry out 2 hours

Results: There is no evidence for occurence of signal peptide CDS. The situation of BBa_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 BBa_K177037

Task 2:

Comment:

Due to some problem which our ampicilline I decided to use vector with resistance to kanamycine instead of previously use pSB1A3


 * Prepare the backbone plasmid pSB1K3 to ligation with COX mitochondrial signal.

20 μl purified plasmid DNA product 1 μl PstI (Fermentas) 1 μl SpeI (Fermentas) 5 μl Buffer Tango (Fermentas) 23 μl MQ water
 * Reaction mixture composition:
 * 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.