Team:UNIPV-Pavia/Calendar/July/settimana4

July, 18th

Digestions of previously purified plasmids were performed for ligations:

Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols. Moreover BBa_R0040-BBa_J61002 plasmid purification and quantification was carried out:

In the afternoon gel electrophoresis was performed.

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As shown in figure, all clones were positive (apart from E13 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the undigested plasmid), so we cut and purified the bands of interest. Cells harbouring E13 were again inoculated in 5 ml LB + Amp and also 1 litre of TBE 1x was prepared.

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.

In the late afternoon and E13 was newly digested with restriction endonucleases for screening:

July, 19th

E13 was newly digested for the subsequent gel extraction.

After electrophoresis, E13 insert was succesfully identified and gel extracted (not shown). Meanwhile, sufficient amount of buffer 1 and buffer 2 for MGZ1 competent cell preparation protocol were prepared. E13 DNA was then quantified:

After E13 digestion (E-P), its ligation was performed in a final volume of 10 μl:

Ligations were incubated ON at 16°C.

250 ml of M9 were prepared, according to protocols.

July, 20th

E24, E25, E26, E27 and were transformed in 100 μl of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35, E36 and BBa_B0032 (transformation efficiency positive control) were transformed in 100 μl of MGZ1 competent cells according to protocols. Plates were incubated ON at 37°C.

In the afternoon 500 ml of LB with chloramphenicol 12.5 were prepared.

July, 21th

All plates showed a lot of colonies, except for E28 and E31 (2 colonies).

A 26x mix was prepared in order to perform PCR on E24-1, E24-2, E25-1, E25-2, E26-1, E26-2, E27-1, E27-2, E28-1, E28-2, E31-1, E31-2, E32-1, E32-2, E33-1, E33-2, E34-1, E34-2, E35-1, E35-2, E36-1, E36-2:

25 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:

• 94°C 30 seconds (denaturing)
• 60°C 1 minute (annealing)
• 72°C 2 minutes (elongation)

dubbio: diciamo dei sequenziamenti?!!

After the PCR, two medium size agarose gels were prepared in order to carry out DNA samples:

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All the band lengths were correct, except E31-1; 750 μl of E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2, E34-1 and E35-2 liquid cultures were used to prepare glycerol stocks.

July, 22th

E36 was transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C. E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2 and E34-1 plasmid purification was carried out, in order to prepare samples for sequencing.

parte sequenziamenti