Team:UNIPV-Pavia/Calendar/October/settimana1

OCTOBER: WEEK 1

I74 massive PCR screening.

As you can see we had the same results as previous PCR. We decided to digest I74-7/8 (two different genotypes) but during centrifugation step for mini we discovered a red pellet in the first one; so we threw it away.

Miniprep and quantification of:

• INTEIN_C3: 69 ng/ul
• I74-8: 78,2 ng/ul
• I54: 130,9 ng/ul
• I35: 69 ng/ul

Digest:

• INTEIN_C3: E-X
• I74-8: E-S (it was also a screening after not clear PCR results)
• I54: E-X
• I35: E-P

Gel run/cut/quantification:

Digest.

I54 wasn't in gel. ???

• INTEIN_C3 (E-X):
• I35 (E-P):
• I74-8 (E-S)

Ligations:

• I78: I74 (E-S) + INTEIN_C3 (E-X)

Transormation (1ul) into E. coli TOP10 of:

• I75
• I77

They were plated on LB+Amp agar plates and let grow ON at +37°C, 220 rpm.

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid

October, 5th

I75 and I77 plates showed lots of red colonies. It's unexplainable, nothing should have carried RFPs or something similiar. Probably we had a kind of contamination during digestion or ligation. So we decided to perform digestions again:

• I55: E-S
• I37: E-X
• I57: E-X
• I31: E-S

and we added

• I54: (E-X)

Digestions were let at +37°C ON.

Transformation (1ul) of

• I78: I74 (E-S) + INTEIN_C3 (E-X)

into E. coli TOP10. In order to check our E. coli TOP10 competent cells we plated them on LB+Amp and Cm agar plates to see if something grew.

ON ligation of:

• I79: I60 (E-S) + INTEIN_C3 (E-X)

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid

October, 6th

E. coli TOP10 plated on LB+Amp agar plate showed red colonies!!! We will perform competentization again ASAP (competent cells were conteminated, not DNA !!!).

Transformation of available ligations I75 and I77 (1ul) into E. coli TOP10, I79 into E. coli STBL3. They were plated on LB+Amp agar plates and let grow ON at +37°C, 220 rpm.

Gel run and cut for ON digestions:

Digest (I31, I37, I57 weren't necessary anymore).

and Nanodrop quantification:

• I54 (E-X): 26 ng/ul
• I55 (E-S): 10 ng/ul

We finally performed ON ligation

• I76: I55 (E-S) + I54 (E-X)

Colony PCR screening for I78 (eleven colonies were picked from LB+Cm agar plate and were inoculated into 5 ml LB+Cm34 and let grow ON for stock). PCR was performed this day but it would have been run the following one (too late this evening).

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid

October, 7th

I78 screening gel run

Screening PCR for I78 (we cat blank from this picture but it was very clear, trust me).

We had extraband in all samples (similar result in I74 PCR: after digest we had a confirmation that it was right. Probably here it was the same problem: very similar repeated sequences give strange PCR; however we sent it sequencing).

Glycerol stock and miniprep for

• I78-3: 153,8 ng/ul

Colony PCR for screening of I79 (eleven colonies were picked).

Screening PCR for I79.

Same as the previous PCR. We took I79-2 that was inoculated into LB+Cm34 and let grow ON at +37°C, 220 rpm.

Transformation (1ul) of

• I0_1C3
• I3_1C3
• I7_1C3
• I8_1C3
• I9_1C3
• I12_1C3
• I76

into E. coli DH5-alpha.

They were plated on LB+Cm34 agar plates (except for I76 that was plated on LB+Amp agar plate) and let grow ON at +37°C.

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid

October, 8th

I76 agar plate showed two/three colonies. It was stored at +4°C; colonies would have been screened the following week.

Glycerol stock, miniprep and Nanodrop quantification of:

• I79-2: 95,3 ng/ul

This DNA (and that from I55 and I78 too) was sent to BMR Genomics for sequencing.

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid

October, 9th

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid

A new part was cloned with these ones, named I80. It is the basic part to build self-inducible promoters and was obtained with the following ligation:

I80=I3(in <partinfo>pSB1C3</partinfo>) (S-P) + <partinfo>BBa_F2620</partinfo> (X-P)

October, 10th

Inoculum into 4 ml LB+Amp of:

• I75-1/2/3
• I76-1/2
• I77-1/2/3

for E-P screening;

• I47
• I48
• I49

for transfer into <partinfo>pSB1C3</partinfo>.

They were let grow at +37°C, 220 rpm.

Transformation of I80 ligation. It was plated on LB+Cm34 agar plate and let grow ON at +37°C.

We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid