Team:UNIPV-Pavia/Calendar/July/settimana3

July, 11th

Digestions of previously purified plasmids were performed for ligations:

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

Medium size gel

Medium size gel

As shown, in figure all clones were positive, so we cut and purified the bands of interest.

After gel extraction, cut DNA was quantified:

Then ligations were performed in a final volume of 10 μl:

Ligations were incubated ON at 16°C.

July, 12th

E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, 19, E20, E21, E22 and E23 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.

July, 13th

All plates showed a lot of colonies, except for E13, E16 (one colony),while for E14, E15 no colony was observed. Because of RFP, E21 was red, E22 and E23 a little bit less. Two colonies for E9, E10, E11, E12, E17, E18, E19, E20, E21, E22, E23 plates were picked. 500 ml of LB with Ampicillin were prepared. ???????????glicerolo stock. diciamo dei reinoculi????????????

July, 14th

A glycerol stock for E21-2, E23-2. E11-1 was prepared. Cultures grew overnight; plasmid purification and quantification were carried out:

For E9-1, E9-2, E10-1, E10-2, E11-1, E11-2, E12-1, E12-2, E17-1, E18-2, E19-1, E19-2, E21-1, E20-2, E21-1, E21-2, E22-1, E22-2, E23-1, E23-2 PCR was performed. ??????????????????????????????????primer? da scrivere???????????????????????????????????????

In order to screen the DNA, a digestion for each ligation was performed.

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

link alle 2 img dei gel con ling wiki

As shown in figure, all clones were positive, except for E9-1,E17-2, E18-1, E18-2, E19-1 which didn' t show any DNA band. E9-2, E17-2, E18-2, E19-2, E20-2, E21-1, E22-2, E23-1 purification was carried out for sequencing.

Cells harbouring E9-2, E10-1, E11-1, E12-1, E13, E16, E21-1, E22-2, E23-1 were inoculated in???? 5 ml???? LB + Amp.

July, 15th

All plates showed a lot of colonies, but the plate containing MGZ1 transformed with BBa_B0032 also showed a lot of satellities.

In order to screen the DNA, digestionS for E17-1, E17-2, E18-1, E18-2 ligation were performed.