Team:UNIPV-Pavia/Calendar/August/settimana3

From 2010.igem.org

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<html><p align="center"><font size="4"><b>AUGUST: WEEK 3</b></font></p></html><hr><br>
<html><p align="center"><font size="4"><b>AUGUST: WEEK 3</b></font></p></html><hr><br>
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<html><a name="indice"/></html>
==August, 16th==
==August, 16th==
Resuspension  from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> (Plate 1, Well 13B) and <partinfo>BBa_I13522</partinfo> (Plate 2, Well 8A).
Resuspension  from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> (Plate 1, Well 13B) and <partinfo>BBa_I13522</partinfo> (Plate 2, Well 8A).
Line 39: Line 39:
Transfomants were plated on LB+Amp 100 ug/ml agar plates, the last one on LB+Amp100+Cm12,5 agar plates and were let grow ON at 37°C.
Transfomants were plated on LB+Amp 100 ug/ml agar plates, the last one on LB+Amp100+Cm12,5 agar plates and were let grow ON at 37°C.
 +
<div align="right"><small>[[#indice|^top]]</small></div>
==August, 17th==
==August, 17th==
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----
----
-
Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:
+
Cultures were diluted (5ul in 2ml LB+antibiotic) for <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test17agosto|Tecan Test]]</font>:
{| border='1' align='center'
{| border='1' align='center'
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Glycerol stock was prepared for ENTERO4C5-T9002.
Glycerol stock was prepared for ENTERO4C5-T9002.
 +
 +
----
 +
 +
<div align="right"><small>[[#indice|^top]]</small></div>
==August, 18th==
==August, 18th==
-
Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:
+
Cultures were diluted (5ul in 2ml LB+antibiotic) for <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test18agosto|Tecan Test]]</font>:
{| border='1' align='center'
{| border='1' align='center'
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Glycerol stock was prepared for I154C5-T9002.
Glycerol stock was prepared for I154C5-T9002.
 +
 +
----
 +
The following cultures were miniprepped and quantified with Nanodrop:
 +
*I26: 67,5 ng/ul
 +
*<partinfo>BBa_K105012</partinfo>: 60,9 ng/ul
 +
*<partinfo>BBa_B0034</partinfo>: 64,5 ng/ul
 +
*<partinfo>BBa_F2620</partinfo>: 91,8 ng/ul
 +
*I20M-1: 191,8 ng/ul
 +
*I20A-1: 176,2 ng/ul
 +
*I21M-3: 156,2 ng/ul
 +
*I21-4: 76,3 ng/ul
 +
*I0: 121,2 ng/ul
 +
*INTEIN: 143,9 ng/ul
 +
*<partinfo>J13002</partinfo>: 31,9 ng/ul
 +
 +
and than digested as follows for four hours:
 +
{| border="1" align='center'
 +
|  ''Culture''    ||  ''Kind''  ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||  ''Enzyme 1 (ul)''  ||  ''Enzyme 2 (ul)'' ||  ''Buffer H (ul)''
 +
|-
 +
|    I26  ||  Insert || 25 ||  21,5  ||  0  ||    0,5 EcoRI  || 0,5 SpeI  || 2,5
 +
|-
 +
|    <partinfo>BBa_K105012</partinfo>  ||  Vector || 25 ||  16,5  ||  5  ||      0,5 E  || 0,5 S  || 2,5
 +
|-
 +
|    <partinfo>BB_B0034</partinfo>  || Vector || 25 ||  15,5  || 6  ||    0,5 E  || 0,5 S    || 2,5
 +
|-
 +
|  <partinfo>BB_F2620</partinfo>  ||  Insert || 25 ||  16,5  ||  5  || 0,5 E  || 0,5 S  || 2,5
 +
|-
 +
|    I20M-1  ||  Insert/Screening || 25 ||  8  ||  13,5  ||  0,5 E  || 0,5 S  || 2,5
 +
|-
 +
|    I20M-1  ||  Insert/Screening || 25 ||  8  ||  13,5  ||  0,5 XbaI || 0,5 PstI  || 2,5
 +
|-
 +
|    I20A-1  ||  Insert/Screening || 25 ||  13  ||  8,5  ||  0,5 E  || 0,5 S  || 2,5
 +
|-
 +
|    I20A-1  ||  Insert/Screening || 25 ||  13  ||  8,5  ||  0,5 X  || 0,5 P  || 2,5
 +
|-
 +
|    I21-4  ||  Insert/Screening || 25 ||  13  ||  8,5  ||  0,5 E  || 0,5 S  || 2,5
 +
|-
 +
|    I21-4  ||  Vector || 25 ||  13,1  ||  8,4  ||  0,5 E  || 0,5 X  || 2,5
 +
|-
 +
|    I0  ||  Vector || 25 ||  8,2  ||  13,3  || 0,5 E  || 0,5 XbaI  || 2,5
 +
|-
 +
|    INTEIN  ||  Insert || 25 ||  10,5  ||  11  || 0,5 E  || 0,5 S  || 2,5
 +
|-
 +
|  <partinfo>BB_J13002</partinfo>  ||  Vector || 25 ||  21,5  ||  0  ||  0,5 S  || 0,5 P  || 2,5
 +
|-
 +
|}
 +
Two medium gel were prepared and samples were loaded and gel run/cut.
 +
 +
{|align="center"
 +
|[[Image:UNIPV10_18_8_10_vettori_fasine.jpg|thumb|300px|center|Vectors digested and gel run/cut]] || [[Image:UNIPV10_18_08_10insertifasine.jpg|thumb|300px|center|Inserts digested and gel run/cut]]
 +
|}
 +
 +
All samples were correctly digested and run; both I20M-1 and I20A-1 are positive, so we decided to use I20M-1; this new screening for I21-4 showed again that it's correct.
 +
 +
Gel extraction gave these results:
 +
*I0 (E-X): 20,8 ng/ul
 +
*<partinfo>BB_F2620</partinfo> (E-S): 13,5 ng/ul
 +
*INTEIN (E-S): 6,5 ng/ul
 +
*<partinfo>BB_B0034</partinfo> (E-X): 23,2 ng/ul
 +
*I20M-1 (E-S): 7,4 ng/ul
 +
*<partinfo>BB_J13002</partinfo> (S-P): 12,5 ng/ul
 +
*I21-4 (E-S): 7,2 ng/ul
 +
*I21-4 (E-X): 24,7 ng/ul
 +
*<partinfo>BBa_K105012</partinfo> (E-X): 23,1 ng/ul
 +
*I26 (E-S): 3,4 ng/ul
 +
*I20M-1 (X-P): 6,5 ng/ul
 +
They were stored ON at -20°C.
 +
 +
----
 +
 +
<div align="right"><small>[[#indice|^top]]</small></div>
==August, 19th==
==August, 19th==
 +
Extra E-X digestion (four hours) of previously miniprepped INTEIN
 +
{|align="center" border="1"
 +
|'''Culture'''||'''Kind'''||'''H2O'''||'''DNA'''||'''Enzyme1'''||'''Enzyme2'''||'''Buffer H'''
 +
|-
 +
|INTEIN || Vector || 13,5 ul||7 ul|| 1 ul EcoRI||1 ul XbaI||2,5 ul
 +
|-
 +
|}
 +
Sample was loaded into a small agarose gel, run and cut.
 +
 +
After gel estraction was quantified with Nanodrop: 19,6 ng/ul.
 +
 +
Finally were performed the following ligations:
 +
*I31: I20M-1 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
 +
*I32: I20M-1 (E-S) + I21-4 (E-X)
 +
*I33: I20M-1 (E-S) + I0 (E-X)
 +
*I34: I21-4 (E-S) + I0 (E-X)
 +
*I35: I21-4 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
 +
*I36: I21-4 (E-S) + I21-4 (E-X)
 +
*I37: INTEIN (E-S) + I0 (E-X)
 +
*I38: <partinfo>BBa_F2620</partinfo> + <partinfo>BBa_B0034</partinfo>
 +
*I39: <partinfo>BBa_J13002</partinfo> + I20M-1 (X-P)
 +
*I40: I26 (E-S) + INTEIN (E-X)
 +
*I41: I26 (E-S) + I0 (E-X)
 +
<div align="right"><small>[[#indice|^top]]</small></div>
==August, 20th==
==August, 20th==
 +
Ligations of previous day were transformed (1 ul) into 100 ul of home-made competent cells '''E. coli'' DH5-alpha. They were than plated on LB+Amp agar plates and incubated ON at 37°C.
 +
 +
----
 +
 +
Colony PCR was performed for MyCRIM (9 colonies), 1 blank, 1 negative control (I5(X-S)), 1 positive control (MrGene CRIM from plate).
 +
 +
[[Image:UNIPV10_20_8_2010colonyPCRMyCREAM-AntarctPhosph9colonie_ladder_blank_RING_CRIMMrGene.jpg.jpg|thumb|300px|center|Screening PCR: MyCRIM (first 9 samples), ladder, blank, RING, CRIM from MrGene.]]
 +
 +
 +
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test20agosto|Tecan Test]]</font>
 +
<div align="right"><small>[[#indice|^top]]</small></div>
==August, 21st==
==August, 21st==
 +
Plates with ligations I31..I41 were stored at +4°C. Colonies would have been screened ASAP.
 +
 +
----
 +
 +
Glycerol stocks and miniprep for MyCrim 1:9.
 +
Quantifications:
 +
{|align="center" border="1"
 +
|'''Culture'''||'''Quantification (ng/ul)'''
 +
|-
 +
|MyCRIM 1|| 59
 +
|-
 +
|MyCRIM 2|| 145
 +
|-
 +
|MyCRIM 3|| 108
 +
|-
 +
|MyCRIM 4|| 125
 +
|-
 +
|MyCRIM 5|| 65
 +
|-
 +
|MyCRIM 6|| 72
 +
|-
 +
|MyCRIM 7|| 97
 +
|-
 +
|MyCRIM 8|| 127
 +
|-
 +
|MyCRIM 9|| 151
 +
|-
 +
|}
 +
 +
Digestions were preformed to screen the direction of the fragment CAT-TT-ORI:
 +
{|align="center" border="1"
 +
|'''DNA''' || '''Buffer B'''|| '''H2O''' || '''EcoRI''' || '''HindIII'''
 +
|-
 +
| 2 ul || 2.5 ul || 18.5 ul || 1 ul || 1 ul
 +
|-
 +
|}
 +
 +
 +
[[Image:UNIPV10_221agostoMyCRIMEco-Hind.jpg|thumb|300px|center|Screening digestions: MyCRIM (from 1 to 9).]]
 +
 +
 +
The fragments length confirm us that all the samples has the CAT-TT-ORI fragment ligated in reversed way.
-
==August, 22nd==
 
 +
<div align="right"><small>[[#indice|^top]]</small></div>
<!-- table previous next week -->
<!-- table previous next week -->
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<tr>
<tr>
<td align="left">[[Team:UNIPV-Pavia/Calendar/August/settimana2| Previous week]]</td>
<td align="left">[[Team:UNIPV-Pavia/Calendar/August/settimana2| Previous week]]</td>
-
<td align="right">Next week</td>
+
<td align="right">[[Team:UNIPV-Pavia/Calendar/August/settimana4| Next week]]</td>
</tr>
</tr>
</table>
</table>

Latest revision as of 15:41, 7 October 2010
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AUGUST: WEEK 3


August, 16th

Resuspension from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> (Plate 1, Well 13B) and <partinfo>BBa_I13522</partinfo> (Plate 2, Well 8A).

Transformation (1 ul) of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM (Mr.Gene) into 100 ul of E. coli TOP10 home-made competent cells.

Transformation of 15-4C5 (1 ul) into in re-competentized E. coli TOP10 carrying <partinfo>BBa_T9002</partinfo>.

Transfomants were plated on LB+Amp 100 ug/ml agar plates, the last one on LB+Amp100+Cm12,5 agar plates and were let grow ON at 37°C.

August, 17th

All plates showed colonies.

Pick of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM, co-trasformed <partinfo>BBa_T9002</partinfo> and inoculum into 1 ml of LB+Amp, LB+Kan, LB+Cm, respectively, to make glycerol stocks. Falcons were than refilled for the minipreps of the following day.

Inoculum of I5, I26, <partinfo>BBa_K105012</partinfo>, <partinfo>BBa_B0034</partinfo>, I20M-1, I20A-1, I21M-3, I21-4, I21A-7, I0 for minipreps of the following day.

Cultures were diluted (5ul in 2ml LB+antibiotic) for Tecan Test:

I7-A I8-5 D I9-1
I10-B I12-2 A2
<partinfo>BBa_B0032</partinfo> I144C5-T9002 I164C5-T9002
I174C5-T9002 I184C5-T9002 I194C5-T9002
ENTERO4C5-T9002

Glycerol stock was prepared for ENTERO4C5-T9002.

August, 18th

Cultures were diluted (5ul in 2ml LB+antibiotic) for Tecan Test:

I7-A I8-5 D I9-1
I10-B I12-2 A2
<partinfo>BBa_B0032</partinfo> I144C5-T9002 I154C5-T9002
I164C5-T9002 I174C5-T9002 I184C5-T9002
I194C5-T9002 ENTERO4C5-T9002

Glycerol stock was prepared for I154C5-T9002.

The following cultures were miniprepped and quantified with Nanodrop:

  • I26: 67,5 ng/ul
  • <partinfo>BBa_K105012</partinfo>: 60,9 ng/ul
  • <partinfo>BBa_B0034</partinfo>: 64,5 ng/ul
  • <partinfo>BBa_F2620</partinfo>: 91,8 ng/ul
  • I20M-1: 191,8 ng/ul
  • I20A-1: 176,2 ng/ul
  • I21M-3: 156,2 ng/ul
  • I21-4: 76,3 ng/ul
  • I0: 121,2 ng/ul
  • INTEIN: 143,9 ng/ul
  • <partinfo>J13002</partinfo>: 31,9 ng/ul

and than digested as follows for four hours:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
I26 Insert 25 21,5 0 0,5 EcoRI 0,5 SpeI 2,5
<partinfo>BBa_K105012</partinfo> Vector 25 16,5 5 0,5 E 0,5 S 2,5
<partinfo>BB_B0034</partinfo> Vector 25 15,5 6 0,5 E 0,5 S 2,5
<partinfo>BB_F2620</partinfo> Insert 25 16,5 5 0,5 E 0,5 S 2,5
I20M-1 Insert/Screening 25 8 13,5 0,5 E 0,5 S 2,5
I20M-1 Insert/Screening 25 8 13,5 0,5 XbaI 0,5 PstI 2,5
I20A-1 Insert/Screening 25 13 8,5 0,5 E 0,5 S 2,5
I20A-1 Insert/Screening 25 13 8,5 0,5 X 0,5 P 2,5
I21-4 Insert/Screening 25 13 8,5 0,5 E 0,5 S 2,5
I21-4 Vector 25 13,1 8,4 0,5 E 0,5 X 2,5
I0 Vector 25 8,2 13,3 0,5 E 0,5 XbaI 2,5
INTEIN Insert 25 10,5 11 0,5 E 0,5 S 2,5
<partinfo>BB_J13002</partinfo> Vector 25 21,5 0 0,5 S 0,5 P 2,5

Two medium gel were prepared and samples were loaded and gel run/cut.

Vectors digested and gel run/cut
Inserts digested and gel run/cut

All samples were correctly digested and run; both I20M-1 and I20A-1 are positive, so we decided to use I20M-1; this new screening for I21-4 showed again that it's correct.

Gel extraction gave these results:

  • I0 (E-X): 20,8 ng/ul
  • <partinfo>BB_F2620</partinfo> (E-S): 13,5 ng/ul
  • INTEIN (E-S): 6,5 ng/ul
  • <partinfo>BB_B0034</partinfo> (E-X): 23,2 ng/ul
  • I20M-1 (E-S): 7,4 ng/ul
  • <partinfo>BB_J13002</partinfo> (S-P): 12,5 ng/ul
  • I21-4 (E-S): 7,2 ng/ul
  • I21-4 (E-X): 24,7 ng/ul
  • <partinfo>BBa_K105012</partinfo> (E-X): 23,1 ng/ul
  • I26 (E-S): 3,4 ng/ul
  • I20M-1 (X-P): 6,5 ng/ul

They were stored ON at -20°C.

August, 19th

Extra E-X digestion (four hours) of previously miniprepped INTEIN

CultureKindH2ODNAEnzyme1Enzyme2Buffer H
INTEIN Vector 13,5 ul7 ul 1 ul EcoRI1 ul XbaI2,5 ul

Sample was loaded into a small agarose gel, run and cut.

After gel estraction was quantified with Nanodrop: 19,6 ng/ul.

Finally were performed the following ligations:

  • I31: I20M-1 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
  • I32: I20M-1 (E-S) + I21-4 (E-X)
  • I33: I20M-1 (E-S) + I0 (E-X)
  • I34: I21-4 (E-S) + I0 (E-X)
  • I35: I21-4 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
  • I36: I21-4 (E-S) + I21-4 (E-X)
  • I37: INTEIN (E-S) + I0 (E-X)
  • I38: <partinfo>BBa_F2620</partinfo> + <partinfo>BBa_B0034</partinfo>
  • I39: <partinfo>BBa_J13002</partinfo> + I20M-1 (X-P)
  • I40: I26 (E-S) + INTEIN (E-X)
  • I41: I26 (E-S) + I0 (E-X)

August, 20th

Ligations of previous day were transformed (1 ul) into 100 ul of home-made competent cells 'E. coli DH5-alpha. They were than plated on LB+Amp agar plates and incubated ON at 37°C.

Colony PCR was performed for MyCRIM (9 colonies), 1 blank, 1 negative control (I5(X-S)), 1 positive control (MrGene CRIM from plate).

Screening PCR: MyCRIM (first 9 samples), ladder, blank, RING, CRIM from MrGene.


Tecan Test

August, 21st

Plates with ligations I31..I41 were stored at +4°C. Colonies would have been screened ASAP.

Glycerol stocks and miniprep for MyCrim 1:9. Quantifications:

CultureQuantification (ng/ul)
MyCRIM 1 59
MyCRIM 2 145
MyCRIM 3 108
MyCRIM 4 125
MyCRIM 5 65
MyCRIM 6 72
MyCRIM 7 97
MyCRIM 8 127
MyCRIM 9 151

Digestions were preformed to screen the direction of the fragment CAT-TT-ORI:

DNA Buffer B H2O EcoRI HindIII
2 ul 2.5 ul 18.5 ul 1 ul 1 ul


Screening digestions: MyCRIM (from 1 to 9).


The fragments length confirm us that all the samples has the CAT-TT-ORI fragment ligated in reversed way.




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