Team:UNIPV-Pavia/Calendar/September/settimana3

SEPTEMBER: WEEK 3

September, 14th

Finally Mr.Gene sent us our YEAST: a little difficult to pick up the package but at the end we succeeded!

I52, I54, I55, I56, I57, I58 plates showed in general few colonies; I52 and I5X showed very few colonies (<=5). Very strange (mumble mumble...), they will be screened ASAP (stored at +4°C).

Miniprep and quanfification of:

• I26: 69,7 ng/ul
• I31: 89,5 ng/ul
• I34: 147,6 ng/ul

3-hours digestion:

• I26: XbaI-PstI (Insert)
• I31: EcoRI-SpeI (Insert)
• I34: E-X (Vector)

Gel run/cut of samples (I26 and I31 insert bands were very very soft)

Gel run/cut for digested I26, I31 and I34.

and gel extraction:

• I26 (X-P): 2,6 ng/ul
• I31 (E-S): 1,4 ng/ul
• I34 (E-X): ng/ul

We already had digested DNA so we could perform new ON ligations

• I59: I21 (E-S) + I34 (E-X)
• I60: I20 (S-P) + I26 (X-P)
• I61: I31 (E-S) + I37 (E-X)

and repeat

• I53: I32 (E-S) + I37 (E-X)

Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN test of the following day.

Screening PCR on 3 colonies picked from MC42 and MC43 respectively. Two method were used:

September, 15th

I47, I48, I49 cultures were diluted 1:100 into 5ml LB+Amp. In the afternoon all cultures were synchronized to O.D. 0,02 and TECAN test was started to check GFP production by these BioBricks.

Transformation of I 53, I59, I60, I61 ligations into E. coli DH5-alpha. They were plated on LB+Amp agar plates and let grow ON at 37°C.

September, 16th

Screening through "colony PCR" for ligations:

• I52
• I53
• I54
• I55
• I56
• I57
• I58
• I59
• I60
• I61

For each plate we picked X colonies that were also inoculated into 1 ml LB+Amp in order to be ready to make glycerol stock of positive ones.

Agarose gel was prepared and samples loaded and run:

Gel run for ligations amplified through PCR.

As you can see ...

September, 17th

September, 18th