Team:UNIPV-Pavia/Calendar/September/settimana4

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SEPTEMBER: WEEK 4



September, 20th

Screening PCR on 6 colonies picked from I55 was performed. The results were the follow:

I55.

Inoculum of I52, I53, I55, I56 and I57 in LB+Amp for MiniPrep of the following day

September, 21st

Miniprep and Nanodrop quantification for:

  • I52 : 92,8 ng/ul
  • I53 : 118,5 ng/ul
  • I55 : 121,1 ng/ul
  • I56 : 159,6 ng/ul
  • I57 : 103 ng/ul

For I61 the DNA is already available quantified 147,1 ng/ul.

After 3 hour digestion was performed:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
I52 Vector 25 10,8 9,7 1 EcoRI 1 XbaI 2,5
I53 Vector 25 8,5 12 1 EcoRI 1 XbaI 2,5
I55 Insert 25 14 6,5 1 EcoRI 1 SpeI 2,5
I56 Insert 25 10 10,5 1 XbaI 1 PstI 2,5
I57 Vector 25 9,7 10,8 1 EcoRI 1 XbaI 2,5
I61 Vector 25 6,8 13,7 1 EcoRI 1 XbaI 2,5


Gel run/cut of samples

UNIPV1021 09 10.jpg

As you can see only I52, I56, I57 and I61 run correctly so gel extraction was performed on this samples:

  • I52 = 29,4 ng/ul
  • I56 = 15,4 ng/ul
  • I57 = 24,7 ng/ul
  • I61 = 30,2 ng/ul

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

  • I62 = I59(X-P) + I39(S-P)
  • I63 = I61(E-X) + I38(E-S)
  • I64 = I52(E-X) + I58(E-S)
  • I65 = I39(S-P) + I56(X-P)
  • I66 = I57(E-X) + I60(E-S)

Tecan Test on self-inducible promoters was performed on prepared samples with a different protocol that is to bring the cultures to a known OD.

September, 22nd

Trasformation of ligation of I62, I63, I64, I65 and I66 into E.coli TOP10.

MiniPrep and quantification of:

  • MyYeast1 = 177,8 ng/ul
  • MyYeast2 = 98,9 ng/ul
  • MyYeast3 = 197,2 ng/ul

Screening digestion was performed as follows:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer Tango (ul)
MyYeast1 Screening 25 1,2 19,3 1 EcoRI 1 NsiI 2,5
MyYeast2 Screening 25 2 18,5 1 EcoRI 1 NsiI 2,5
MyYeast3 Screening 25 1 19,5 1 EcoRI 1 NsiI 2,5

Gel run of samples was performed(foto)


Noone samples was positive


Glycerol stocks of MyYeast1, MyYeast2, MyYeast3 was performed.

Inoculum in LB+Amp+Cm12,5 from glycerol stocks and colony for antibiotic resistance testing of I47_S1, I48_S1, I49_S1 and I13522.

September, 23rd

Digestion of yesterday are repeted (because noone of them was postitive) as follows:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme (ul) Buffer H Roche 10X (ul)
MyYeast1 Screening 25 1,2 20,3 1 EcoRI 2,5
MyYeast2 Screening 25 2 19,5 1 EcoRI 2,5
MyYeast3 Screening 25 1 20,5 1 EcoRI 2,5


Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme (ul) Buffer Red (ul)
MyYeast1 Screening 25 1,2 20,3 1 NsiI 2,5
MyYeast2 Screening 25 2 19,5 1 NsiI 2,5
MyYeast3 Screening 25 1 20,5 1 NsiI 2,5


Gel run of samples was performed (foto)

All samples seem to be positive!!


Inoculum of I62, I63, I64, I65 and I66 of 3 colonies in LB+Amp 5ml.

September, 24th

Glycerol stocks of

  • I62-1,2,3
  • I63-1,2,3
  • I64-1,2,3
  • I65-1,2,3
  • I66-1,2,3


September, 25th