Team:UNIPV-Pavia/Calendar/September/settimana5

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Revision as of 10:00, 30 September 2010

SEPTEMBER: WEEK 4

September, 27th

MiniPrep for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f

(foto)

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

DNA pcr for MyYeast 1-2-3

Protein electrophoresis for phasins (Fede)

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September, 28th

Tecan Test Giacomo

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September, 29th

Tecan Test Giacomo

Colony PCR for 6 newly picked colonies: no positives nor this time.

Gel run for all digested parts 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)

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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

(foto)

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>pSB1C3</partinfo>

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

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October, 1st

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October, 2nd

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@@ Line 58: / Line 58: @@
 ----
-Miniprep and quantification for:
+Colony PCR for 6 newly picked colonies: no positives nor this time.
-*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)
 Gel run for all digested parts and gel run/cut/purification for:
@@ Line 82: / Line 70: @@
 *<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul
 *<partinfo>pSB1C3</partinfo>: 10 ng/ul
@@ Line 95: / Line 82: @@
 |I73= I38 (E-S) + I53 (E-X)
 |}