Team:UNIPV-Pavia/Calendar/September/settimana4

From 2010.igem.org

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(→September, 23rd)
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Digestion of yesterday are repeted (because noone of them was postitive) as follows:
Digestion of yesterday are repeted (because noone of them was postitive) as follows:
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{| border="1" align='center'
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|  ''Culture''    ||  ''Kind''  ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||  ''Enzyme (ul)''  ||  ''Buffer H Roche 10X (ul)''
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|-
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|    MyYeast1  ||  Screening || 25 ||  1,2 ||  20,3  ||  1 EcoRI  || 2,5
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|-
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|    MyYeast2  ||  Screening || 25 ||  2 ||  19,5  ||  1 EcoRI  || 2,5
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|-
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|    MyYeast3  ||  Screening || 25 ||  1 ||  20,5  ||  1 EcoRI  || 2,5
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|}
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 +
{| border="1" align='center'
 +
|  ''Culture''    ||  ''Kind''  ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||  ''Enzyme (ul)''  ||  ''Buffer Red (ul)''
 +
|-
 +
|    MyYeast1  ||  Screening || 25 ||  1,2 ||  20,3  ||  1 NsiI  || 2,5
 +
|-
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|    MyYeast2  ||  Screening || 25 ||  2 ||  19,5  ||  1 NsiI  || 2,5
 +
|-
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|    MyYeast3  ||  Screening || 25 ||  1 ||  20,5  ||  1 NsiI  || 2,5
 +
|}
<div align="right"><small>[[#indice|^top]]</small></div>
<div align="right"><small>[[#indice|^top]]</small></div>

Revision as of 14:32, 24 September 2010
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Week 1

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Week 4

Week 5


SEPTEMBER: WEEK 4


September, 20th

Screening PCR on 6 colonies picked from I55 was performed. The results were the follow:

I55.

Inoculum of I52, I53, I55, I56 and I57 in LB+Amp for MiniPrep of the following day

September, 21st

Miniprep and Nanodrop quantification for:

  • I52 : 92,8 ng/ul
  • I53 : 118,5 ng/ul
  • I55 : 121,1 ng/ul
  • I56 : 159,6 ng/ul
  • I57 : 103 ng/ul

For I61 the DNA is already available quantified 147,1 ng/ul.

After 3 hour digestion was performed:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
I52 Screening 25 10,8 9,7 1 EcoRI 1 XbaI 2,5
I53 Screening 25 8,5 12 1 EcoRI 1 XbaI 2,5
I55 Screening 25 14 6,5 1 EcoRI 1 SpeI 2,5
I56 Screening 25 10 10,5 1 XbaI 1 PstI 2,5
I57 Screening 25 9,7 10,8 1 EcoRI 1 XbaI 2,5
I61 Screening 25 6,8 13,7 1 EcoRI 1 XbaI 2,5


Gel run/cut of samples

UNIPV1021 09 10.jpg

As you can see only I52, I56, I57 and I61 are positive so gel extraction was performed on this samples:

  • I52 = 29,4 ng/ul
  • I56 = 15,4 ng/ul
  • I57 = 24,7 ng/ul
  • I61 = 30,2 ng/ul

We already had digested DNA so we could perform new ON ligations:

  • I62 = I59(X-P) + I39(S-P)
  • I63 = I61(E-X) + I38(E-S)
  • I64 = I52(E-X) + I58(E-S)
  • I65 = I39(S-P) + I56(X-P)
  • I66 = I57(E-X) + I60(E-S)

Tecan Test on self-inducible promoters was performed on prepared samples with a different protocol that is to bring the cultures to a known OD.

September, 22nd

Trasformation of ligation of I62, I63, I64, I65 and I66 into E.coli TOP10.

MiniPrep and quantification of:

  • MyYeast1 = 177,8 ng/ul
  • MyYeast2 = 98,9 ng/ul
  • MyYeast3 = 197,2 ng/ul

Screening digestion was performed as follows:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer Tango (ul)
MyYeast1 Screening 25 1,2 19,3 1 EcoRI 1 NsiI 2,5
MyYeast2 Screening 25 2 18,5 1 EcoRI 1 NsiI 2,5
MyYeast3 Screening 25 1 19,5 1 EcoRI 1 NsiI 2,5

Gel run of samples was performed


Noone samples was positive

Glycerol stocks of MyYeast1, MyYeast2, MyYeast3 was performed.

Inoculum in LB+Amp+Cm12,5 from glycerol stocks and colony for antibiotic resistance testing of I47_S1, I48_S1, I49_S1 and I13522.

September, 23rd

Digestion of yesterday are repeted (because noone of them was postitive) as follows:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme (ul) Buffer H Roche 10X (ul)
MyYeast1 Screening 25 1,2 20,3 1 EcoRI 2,5
MyYeast2 Screening 25 2 19,5 1 EcoRI 2,5
MyYeast3 Screening 25 1 20,5 1 EcoRI 2,5
Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme (ul) Buffer Red (ul)
MyYeast1 Screening 25 1,2 20,3 1 NsiI 2,5
MyYeast2 Screening 25 2 19,5 1 NsiI 2,5
MyYeast3 Screening 25 1 20,5 1 NsiI 2,5

September, 24th

September, 25th



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