Team:UNIPV-Pavia/Calendar/September/settimana4

SEPTEMBER: WEEK 4

I55-1..6.

I55-3 seems to be ok.

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:

Gel run/cut of samples

I52(E-X), I53(E-X), I55(E-S), I56(X-P), I57(E-X), I61(E-X).

As you can see only I52, I56, I57 and I61 run correctly (I55 was wrong) so gel extraction was performed on these 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 = I39(S-P) + I59(X-P)
• I63 = I38(E-S) + I61(E-X)
• I64 = I58(E-S) + I52(E-X)
• I65 = I39(S-P) + I56(X-P)
• I66 = I60(E-S) + I57(E-X)

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 ligations 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:

Gel run of samples was performed

MyYeast-1/2/3 (EcoRI-NsiI digest).

None of 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 none of them were postitive) as follows:

Gel run of samples was performed

MyYeast-1/2/3 digest.

All samples seem to be positive!!

Inoculum for three colonies from I62, I63, I64, I65 agar plates into 5ml LB+Amp.

September, 24th

Glycerol stocks was performed for

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

MiniPrep and Nanodrop quantification :

• I62-1 : 35,1 ng/ul
• I62-2 : 27,8 ng/ul
• I62-3 : 63,8 ng/ul
• I63-1 : 195,3 ng/ul
• I63-2 : 68,3 ng/ul
• I63-3 : 148,7 ng/ul
• I64-1 : 97,8 ng/ul
• I64-2 : 171,8 ng/ul
• I64-3 : 146,3 ng/ul
• I65-1 : 69,7 ng/ul
• I65-2 : 44 ng/ul
• I65-3 : 294,3 ng/ul
• I66-1 : 292,8 ng/ul
• I66-2 : 198 ng/ul
• I66-3 : 284,8 ng/ul

Digestion for screening :

• I62-1,2,3 (E-P)
• I63-1,2,3 (E-P)
• I64-1,2,3 (E-P)
• I65-1,2,3 (E-P)
• I66-1,2,3 (E-P)

Gel run of samples was performed

Screening for I62/3/4/5/6 (E-P) digested.

As you can see I62-1/2/3 were all negative; I63-1/2/3, I64-1/2/3, I65-3, I66-1/2/3 were positive. So we made glycerol stocks for I63-1, I64-2, I65-3, I66-1 and stored them at -80°C. We stored their DNA in -20°C, too.

Dilution 1:50 of I47, I48, I49, GFP in LB+Amp+Cm12,5 and S1 in LB+Cm12,5.

All cultures were grow up to OD=0,1 of Nanodrop. They were then induced with HSL (lysis induction) 100nM

September, 26th

Inoculum of <partinfo>BBa_K173000</partinfo>, I47, I48, I49, <partinfo>BBa_B0031</partinfo> into 3 ml LB+Amp for tomorrow TECAN test. Pick of six colonies from I66 agar plate (I66-A/B/C/D/E/F) and inoculum into 3 ml LB+Amp for screening thorough E-P digest. All colonies were let grow ON at 37°C, 220 rpm.