Team:UNIPV-Pavia/Calendar/June/settimana3

June, 14th

Inoculum in 5ml LB+Amp from glycerol stock for:

• I0-1
• I0-2
• I1-1
• I1-2
• I2-1 (phenotipic screening ok, grown both on LB+Amp and LB+Cm)
• I3-1
• I3-2

Cultures were grown overnight at 37°C 220 rpm.

June, 15th

Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit.

After MiniPrep, purified DNA was quantified with NanoDrop.

Digestion of:

Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.

Two gels were prepared, as shown in figure.

Medium gel

Small gel

Gel results show that:

• I0-1 is negative, while I0-2 is positive: we choose I0-2, from now on named I0, for sequencing and for gel extraction of Insert.
• Both I1-1 and I1-2 are positive. We choose I1-2, from now on named I1, for sequencing and gel-extraction because its DNA concentration is better.
• I2-1, from now on I2, is positive so it will be used for sequencing and gel-extraction.
• I3-2 is negative, while I3-1, from now on I3, is used for sequencing and gel-extraction.

So I0-2, I1-2, I2-1 and I3-1 samples are prepared for sequencing.

Ligation of:

• I4: BBa_K165037 (S-P) + I1 (X-P)
• I5: I2 (E-S) + BBa_J61001 (E-X)
• I6: I3 (E-S) + BBa_T9002 (E-X)

Ligation of I4, I5 and I6 was performed at 16°C overnight.

June, 16th

Ligations I4, I5 and I6 were transformed in E. coli:

• I4 and I6 in E. coli DH5-alpha
• I5 (final part) in E. coli TOP10

1ul of ligation was transformed in 100ul competent cells.

June, 17th

We checked the presence of colonies in plates of I4, I5 and I6 incubated overnight at 37°C. All plates showed colonies. I4 had big, round single colonies. I5 showed big colonies surrounded by small colonies. I6 showed few small colonies and for this reason it was further incubated for 3 hours. Colonies were picked and inoculated in 1ml LB+antibiotic.

I5-1 and I5-2 were inoculated both in LB+Amp and LB+Cm to have a phenotipic assay: in fact I5 should express the Chloramphenicol resistance. All 9 cultures were incubated at 37°C 220 rpm for 6 hours.

250 ml LB and 250ml LB+Cm for low copy plasmids (91,5 ul of Cm from 1000x stock in 250 ml LB).

After six hours, we checked the growth in liquid of our colonies.

• I4-1 and I4-2 were all grown
• I5-1 and I5-2 were both grown on LB+Amp, but only I5-1 was grown in LB+Cm. I5-2 was negative at this phenotipic assay, so it was discarded.
• I6-1 was apparently not grown, so it was discarded. I6-2 and I6-2 were grown, but the cultures were more limpid than the others, showing that I6 has a slower growth than nomal.

Glycerol stocks were prepared for:

and stored at -80°C.

Remaining cultures were re-filled with 5ml LB+Amp and inoculated at 37°C 220rpm for tomorrow MiniPrep.

June, 18th

All cultures incubated at 37°C 220 rpm overnight grew. MiniPrep was performed, with the following quantifications:

Digestion of:

for 1h at 37°C.

Gel run for screening

All the clones are OK :)