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JULY: WEEK 4
July, 18thBBa_R0040 in BBa_J61002 plasmid purification and quantification was carried out:
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.
In the afternoon gel electrophoresis was performed. As shown in figure, all clones were positive (apart from E13-1 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the linearized plasmid), so we cut and purified the bands of interest. After gel extraction, digested DNA was quantified:
Then ligations were performed in a final volume of 10 μl:
Ligations were incubated ON at 16°C.
July, 19th
According to protocols 200 ml of Buffer 1 and 100 ml of Buffer 2 were prepared for MGZ1 strain competentization.
After gel electrophoresis, E13-1 insert was succesfully identified. E13-1 digested DNA was gel-extracted and quantified:
E28 ligation was performed in a final volume of 10 μl:
Ligation was incubated ON at 16°C.
250 ml of M9 were prepared, according to protocols. July, 20th
E24, E25, E26 and E27, E36 were transformed in 100 μl of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35 and BBa_B0032 (4 ng of DNA to test transformation efficiency) were transformed in 100 μl of MGZ1 competent cells according to protocols; a negative control was also plated in order to avoid the presence of contaminants. Plates were incubated ON at 37°C.
July, 21stAll plates showed a lot of colonies, except for E28, E31 (2 colonies) and E36; two colonies for each of them were picked, inoculated and screened by PCR. Positive control plate for MGZ1 showed few colonies due to poor transformation efficiency (new comptetent MGZ1 will be prepared!); no colonies grew on negative control plate. 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:
24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:
Two medium size agarose gels were prepared; in the afternoon gel electrophoresis was carried out:
All the band lengths were correct, except E31-2, E36-1 and E36-2 (E36 was plated on the wrong antibiotic!). July, 22ndE36 was transformed in 100 μl of TOP10 competent cells according to protocols. The plate was incubated at room temperature. E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2 and E34-1 plasmid purification was carried out:
E17-2, E18-2, E28-1, E31-1, E32-2, E33-2, E34-1, E35-2 purified DNA was sent for sequencing to BMR Genomics.
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Team:UNIPV-Pavia/Calendar/July/week4
From 2011.igem.org
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Latest revision as of 15:49, 20 September 2011