Team:UNIPV-Pavia/Calendar/September/settimana5

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Revision as of 09:55, 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.

DNA pcr for MyYeast 1-2-3

ColonyPCR for I55?? (insert: about 1600bp...)

Protein electrophoresis for phasins (Fede)

^top

September, 28th

Tecan Test Giacomo

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September, 29th

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)

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 102: / Line 102: @@
 ==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)
@@ Line 110: / Line 112: @@
 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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