Team:UNIPV-Pavia/Calendar/June/settimana2

From 2010.igem.org

(Difference between revisions)

Latest revision as of 12:12, 22 October 2010

JUNE: WEEK 2

June, 7th

These BioBrick were resuspended in 15ul ddH20:

<partinfo>BBa_E2050</partinfo> (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
<partinfo>BBa_K165018</partinfo> (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
<partinfo>BBa_K165037</partinfo> (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
<partinfo>BBa_P1004</partinfo> (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
<partinfo>BBa_J61001</partinfo> (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
<partinfo>BBa_K081008</partinfo> (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
<partinfo>BBa_K125500</partinfo> (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2

1,5 ul of each culture was transformed in 100 ul of home-made competent E. coli DH5alpha.

Tranformants were all plated on LB agar plates added with Ampicillin, except for <partinfo>BBa_E2050</partinfo> (Kanamycin).

500 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).

^top

June, 8th

All plates grown overnight at 37°C showed colonies! In particular, <partinfo>BBa_E2050</partinfo> (grown on LB+Kan), <partinfo>BBa_K125500</partinfo> (grown on LB+Amp), <partinfo>BBa_K081008</partinfo> (grown on LB+Amp) and <partinfo>BBa_K165018</partinfo> (grown on LB+Amp) showed big colonies, well separated on the plate. <partinfo>BBa_P1004</partinfo> (grown on LB+Amp) and <partinfo>BBa_J61001</partinfo> (grown on LB+Amp) showed many colonies, with big colonies surrounded by small colonies. <partinfo>BBa_K165037</partinfo> (grown on LB+Amp) showed only 11 big colonies.

A colony was picked for each plate and inoculated in 1ml LB+antibiotic.

<partinfo>BBa_E2050</partinfo>	1ml LB+Kan
<partinfo>BBa_K165037</partinfo>	1ml LB+Amp
<partinfo>BBa_P1004</partinfo>	1ml LB+Amp
<partinfo>BBa_K125500</partinfo>	1ml LB+Amp
<partinfo>BBa_K081008</partinfo>	1ml LB+Amp
<partinfo>BBa_J61001</partinfo>	1ml LB+Amp
<partinfo>BBa_K165018</partinfo>	1ml LB+Amp

Sara and Nicolò at work

<partinfo>BBa_K165037</partinfo> in pSB1AK3 plasmid was inocultaed both in 1ml LB+Amp and in 1ml LB+Kan for a phenotipic assay.

Cultures were grown for 8 hours at 37°C 220 rpm.

After this time, cultures were all grown and in saturation phase. <partinfo>BBa_K165037</partinfo> in LB+Kan was also grown, so that this phenotipic assay was positive.

Glycerol stocks were prepared for each culture (250ul saturated culture + 750 ul glycerol 80%) and stored at -80°C.

The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.

^top

June, 9th

Ligation of:

I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X)
I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)

<partinfo>BBa_B0015</partinfo> was accidentally not inoculated yesterday... :( Luckily, we had a DNA stock for this part stored at -20°C, so we used this stock for ligations.

Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit. Surprisingly, after the first centrifugation, we observed that <partinfo>BBa_E2050</partinfo> pellet was orange!! This is curious, because this BioBrick expresses mOrange, but it lacks of promoters. Probably, this spurious transcription is due to the promoter of Kanamycin resistance, that is oriented in the same direction.

BBa_E2050 orange pellet

After MiniPrep, purified DNA was quantified with NanoDrop.

<partinfo>BBa_E2050</partinfo>	139,2 ng/ul
<partinfo>BBa_K165037</partinfo>	181,5 ng/ul
<partinfo>BBa_P1004</partinfo>	132,8 ng/ul
<partinfo>BBa_K125500</partinfo>	124,3 ng/ul
<partinfo>BBa_K081008</partinfo>	138,3 ng/ul
<partinfo>BBa_J61001</partinfo>	122,2 ng/ul
<partinfo>BBa_K165018</partinfo>	166,3 ng/ul

Digestion of:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1	Enzyme 2	Buffer H
<partinfo>BBa_K125500</partinfo>	Insert	25	16	4,5	1 EcoRI	1 SpeI	2,5
<partinfo>BBa_B0015</partinfo>	Vector	25	6,3	14,2	1 EcoRI	1 XbaI	2,5
<partinfo>BBa_E2050</partinfo>	Insert	25	14	6,5	1 EcoRI	1 SpeI	2,5
<partinfo>BBa_K165018</partinfo>	Vector	25	6	14,5	1 EcoRI	1 XbaI	2,5
<partinfo>BBa_P1004</partinfo>	Insert	25	15	5,5	1 EcoRI	1 SpeI	2,5
<partinfo>BBa_K081008</partinfo>	Insert	25	14,5	6	1 EcoRI	1 SpeI	2,5

Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.

Gel run/cut of cultures

Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.

^top

June, 10th

I0, I1, I2 and I3 were tranformed in home made compotent E. coli DH5-alpha and plated on LB+Amp agar plates.

Plates with transformed E.coli were incubated overnight at 37°C 220 rpm.

The four plates were named:

iGEM 2010 10/06/2010 DH5-alpha I0 LB+Amp
iGEM 2010 10/06/2010 DH5-alpha I1 LB+Amp
iGEM 2010 10/06/2010 DH5-alpha I2 LB+Amp
iGEM 2010 10/06/2010 DH5-alpha I3 LB+Amp

and stored at +4°C.

^top

June, 11th

All four plates grown overnight showed colonies!! Two colonies for each plate were picked and incubated in 1ml LB+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:

I0-1 in 1ml LB+Amp;	I0-2 in 1ml LB+Amp
I1-1 in 1ml LB+Amp;	I1-2 in 1ml LB+Amp
I2-1 in 1ml LB+Amp;	I2-2 in 1ml LB+Amp
I2-1 in 1ml LB+Cm;	I2-2 in 1ml LB+Cm
I3-1 in 1ml LB+Amp;	I3-2 in 1ml LB+Amp

After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-2 were grown both in LB+Amp and LB+Cm.

8 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.

^top

@@ Line 7: / Line 7: @@
 {{UNIPV-Pavia/Style}}
 <tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td>
 <td valign="top">
-<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px">
+<table border="0" align="center" width="100%">
-<html><p align="center"><font size="4"><b>JUNE: WEEK 2</b></font></p></html><hr><br>
+<tr><td align="justify" valign="top" style="padding:20px">
+<table class="menu" border="0" width="100%">
+<tr>
+<td align="center" style="padding:0; height:20px">
+[[Team:UNIPV-Pavia/Calendar/June/settimana2|Week 2]]
+</td>
+<td align="center">
+[[Team:UNIPV-Pavia/Calendar/June/settimana3|Week 3]]
+</td>
+<td align="center">
+[[Team:UNIPV-Pavia/Calendar/June/settimana4|Week 4]]
+</td>
+</tr>
+</table>
+<br>
+<html>
+<p align="center"><font size="4"><b>JUNE: WEEK 2</b></font></p></html><hr><br>
+<html><a name="indice"/></html>
 ==June, 7th==
 These BioBrick were resuspended in 15ul ddH20:
@@ Line 27: / Line 50: @@
 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).
+<div align="right"><small>[[#indice|^top]]</small></div>
 ==June, 8th==
-All plates grown overnight at 37°C show colonies!
+All plates grown overnight at 37°C showed colonies!
 In particular, <partinfo>BBa_E2050</partinfo> (grown on LB+Kan), <partinfo>BBa_K125500</partinfo> (grown on LB+Amp), <partinfo>BBa_K081008</partinfo> (grown on LB+Amp) and <partinfo>BBa_K165018</partinfo> (grown on LB+Amp) showed big colonies, well separated on the plate.
 <partinfo>BBa_P1004</partinfo> (grown on LB+Amp) and <partinfo>BBa_J61001</partinfo> (grown on LB+Amp) showed many colonies, with big colonies surrounded by small colonies.
 <partinfo>BBa_K165037</partinfo> (grown on LB+Amp) showed only 11 big colonies.
-A colony was peaked for each plate and inoculated in 1ml LB+antibiotic.
+A colony was picked for each plate and inoculated in 1ml LB+antibiotic.
@@ Line 42: / Line 67: @@
 <tr>
 <td>
-{|
+{|border='1'
 |  <partinfo>BBa_E2050</partinfo>    ||   1ml LB+Kan
 |-
@@ Line 85: / Line 110: @@
 The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.
+<div align="right"><small>[[#indice|^top]]</small></div>
 ==June, 9th==
@@ Line 113: / Line 140: @@
 After MiniPrep, purified DNA was quantified with NanoDrop.
-{|
+{|border='1'
 |  <partinfo>BBa_E2050</partinfo>    ||   139,2 ng/ul
 |-
@@ Line 134: / Line 161: @@
 Digestion of:
-{|
+{|border='1'
 |  ''Culture''    ||   ''Kind''   ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||   ''Enzyme 1''  ||  ''Enzyme 2'' ||   ''Buffer H''
 |-
@@ Line 157: / Line 184: @@
 <br>
 Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.
+<div align="right"><small>[[#indice|^top]]</small></div>
 ==June, 10th==
@@ Line 164: / Line 193: @@
 Plates with transformed ''E.coli'' were incubated overnight at 37°C 220 rpm.
-<table align='center'><tr><td>
-[[Image:UNIPV_attivita_lab4.jpg|thumb|300px|]]
-</td></tr></table>
 The four plates were named:
@@ Line 174: / Line 200: @@
 * iGEM 2010 10/06/2010 DH5-alpha I3 LB+Amp
-and are stored at +4°C.
+and stored at +4°C.
+<div align="right"><small>[[#indice|^top]]</small></div>
 ==June, 11th==
 All four plates grown overnight showed colonies!!
-Two colonies for each plate were peaked and incubated in 1ml LB+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:
+Two colonies for each plate were picked and incubated in 1ml LB+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:
 {|
@@ Line 196: / Line 224: @@
 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.
+<div align="right"><small>[[#indice|^top]]</small></div>
+<!-- table previous next week -->
+<br><br>
+<table border="0" width="100%" class="menu">
+<tr>
+<td align="left">Previous week</td>
+<td align="right">[[Team:UNIPV-Pavia/Calendar/June/settimana3| Next week]]</td>
+</tr>
+</table>
+<!-- fine table -->
+</td>
+<td width="15%" valign="top">
+{{UNIPV-Pavia/menu_mesi}}
+</td>
+</tr>
+</table>