Team:UNIPV-Pavia/Calendar/August/settimana2

AUGUST: WEEK 2

August, 9th

Phasins plates showed very few colonies (12 for I20-new and 17 for I21-new): they were all picked and let grow in LB+Amp 100ug/ml. In the evening we made glycerol stocks and re-filled the tubes for the screening of the following day:

In order not to loose our time we decided to perform again the PCR-amplification/modification of <partinfo>BBa_K208001</partinfo> in case of a negative screening. Than we gel-ran and gel-extracted right amplicons. PCR was performed with:

• 10_F and S_R primers
• S_F and S_R primers

PCR results: PhaP 10-S and PhaP S-S

The image shows results of our PCR: both amplicons are positive, so we purified cut DNA, obtaining the following quantifications:

• PhaP 10-S: 16,8 ng/ul
• PhaP S-S: 17,1 ng/ul

Tomorrow we will proceed to digest this part, in order to have an alternative if all clones of I20 and I21 are negative.

In the afternoon, we inoculated all parts we will need tomorrow.

Check of LB+Cm 6 ug/ml agar plates. We let grow at 30°C:

• MG1655 in LB
• MC1061 in LB
• MG123/008 in LB+Amp 50 ug/ml
• MC123/008 in LB+Amp 50 ug/ml
• F2620-4C5 (positive control)

Than we streaked cultures on a seven-sector divided plate, and we let it grow ON at 30°C. Since 6 ug/ml is a very low concentration, we wanted to check if actually nothing (except F2620-4C5) grow on these plates.

Transformation at 30°C of 100 ul of MG13/008 and MC123/008 competent cells to check their efficiency. We used 1 ul (~4 ng) of RING, F2620-4C5, Nothing (water) and plated on proper LB agar plates:

• RING: Cm 34 ug/ml
• F2620-4C5 (positive control): Cm 12,5 ug/ml
• Nothing (negative control): Cm 12,5 ug/ml

Both RING and F2620-4C5 should survive, but we had a problem so that MC008 transformed with RING couldn't be plated. We will check it another time. We let grow plates ON, 30°C.

August, 10th

Glycerol stock was prepared for:

• linker
• I22-1
• I22-2
• I22-3
• I23-1
• I23-2
• I23-3
• I8-5 D
• I8-5 E
• I8-5 F

and are stored at -80°C.

Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:

MiniPrep was performed for following cultures (using our new NucleoSpin kit, that we prepared yesterday! :) ):

Digestion of:

°(after '+' added after 2 hours and incubated for further 2 hours)

Digestions were incubated at 37°C for 3 hours. A big gel and 2 medium gel were prepared, samples were loaded and gel ran/cut.

Up: Screening for I20-1..12 (PhaP 10-S (X-P)): all colonies are negative :( Down: Screening for I21-1..14 (PhaP S-S (X-P)): all colonies are negative, except for I21-4 gel run/cut and purified (we ran gel again after the cut).

Screening for I21-15..17, I22, I23 and I15-1. The three colonies of I21 were negative, I22 and I23 were positive, so they were excided and purified. I15-1 was excided, even if extra-bands were observed. We use it for ligation because sequencing is ok!

Ent4C5, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_K105012</partinfo> and <partinfo>BBa_R0062</partinfo> were extracted and purified. Also PhaP 10-S (X-S) and PhaP S-S (X-S) were extracted and purified, in order to repeat the ligation with a vector, since one only colony was positive for I21 and no colony was positive for I20. <partinfo>pSB1A2</partinfo> vector cut X-S was also gel extracted and purified.

After purification, digested DNA was quantified as follows:

Colonies transformed with RING, F2620-4C5, Nothing and plated on proper agar plates were still too little to check efficiency of MC123/008 and MG123/008 competent cells. So we let them grow another day and night at 30°C.

August, 11th

August, 12th

August, 13th

August, 14th

August, 15th