Team:UNIPV-Pavia/Calendar/September/settimana5

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SEPTEMBER: WEEK 4



September, 27th

MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f

DNA pcr for MyYeast 1-2-3

Gel run

I62 and MyYeast series screening (I62, blank, MyYeastx,xbis).


I62-e was positive, so we made glycerol stock and stored it at -80°C.


Protein electrophoresis for phasins (Fede)

September, 28th

Tecan Test Giacomo


Miniprep and quantification for:

  • I1: 59,6 ng/ul
  • I4: 109,9 ng/ul
  • I53: 160,3 ng/ul
  • <partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul
  • <partinfo>BBa_J04450</partinfo>-2: 47 ng/ul

ON digestion of:

  • I1 (E-S)
  • I4 (E-S)
  • I53 (E-X)
  • <partinfo>BBa_J04450</partinfo>-1 (E-P)
  • <partinfo>BBa_J04450</partinfo>-2 (E-P)
  • MyYeast-3 (DNA already available) (E-S)
  • <partinfo>pSB4C5</partinfo> (DNA already available) (E-P)


September, 29th

Tecan Test Giacomo


Colony PCR for 6 newly picked colonies.

I55 colony PCR screening (blank blank 1 ..).

No positives nor this time.



Gel run for all digested parts

Digestions (4C5 myYeast3 I4 I53 J04450-2 J04450-1 I1).

and gel run/cut/purification for:

  • 4C5 (E-P): 19,3 ng/ul
  • My Yeast 3 (E-S): 20,8 ng/ul
  • I1: 3,8 ng/ul
  • I4 (E-S): 14,2 ng/ul
  • I53 (E-X): 21,6 ng/ul
  • <partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul
  • <partinfo>pSB1C3</partinfo>: 10 ng/ul


Ligation of:

I70=MyYeast 3(E-S)+I4(E-S)
I71=MyYeast 3(E-S)+I1(E-S)
I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>
I73= I38 (E-S) + I53 (E-X)



September, 30th

Transformation of I73 (1ul) into E. coli TOP10. It was plated on LB+Amp 100 ug/ml agar plate.


Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:

  • I20: (E-X)

Gel run/cut

I20 E-X digest.

We repeated I55 ligation using newly digested vector and already digested I38 (E-S)

Inoculum into 5ml LB+Amp of:

  • I31
  • I35
  • I37
  • I57
  • I60
  • INTEIN
  • 3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)

Cultures were let grow ON at 37°C, 220 rpm.



October, 1st

Transformation of I55 into E. coli DH5-alpha. It was plated on LB+Amp 100 agar plate.


Miniprep and Nanodrop quantification for:

  • I31:
  • I35:
  • I60:

(they were also send sequencing)

  • INTEIN
  • <partinfo>pSB1C3</partinfo>:
  • I37:
  • I53:

Digest:

  • <partinfo>pSB1C3</partinfo>: E-P
  • <partinfo>pSB1C3</partinfo>: E-S
  • <partinfo>pSB1C3</partinfo>: X-P
  • INTEIN: E-P
  • I60: E-S
  • I31: S-P
  • I35: X-P
  • I37: E-X
  • I57: E-X

Gel run/cut/extraction/quantification:

(foto)

  • I60:
  • I37:
  • I57:

They were stored at -20°C

  • I31:
  • I35:

ON ligation of:

  • IXX: I31 (S-P) + I35 (X-P) (in vector <partinfo>pSB1A2</partinfo>)
  • INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)

Pick and inoculum of I73 into 5 ml LB+Amp 100. It was let grow ON at 37°C, 220 rpm.


October, 2nd