|
AUGUST: WEEK 1
August, 1stTwo colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm1 12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:
These plasmids were digested with EcoRI and SpeI restriction enzymes and screened by gel-electrophoresis; a medium-size agarose gel was then prepared.
Insert lengths were all correct so J101-31, J101-E5, J101-E7 were transformed from MiniPrepped DNA in MGZ1 competent cells. E37-2, E38-1, E39-1, E40-2 and E42-1 were inoculated in 5 ml LB + Cm12.5 and grown ON for plasmid extraction. August, 2ndMGZ1 J101-31, J101-E5 and J101-E7 plates were all grown; one colony was picked from each of them and inoculated in LB + Cm12.5. Plasmids harboring E28-AGAIN-1, E28-AGAIN-2, E41-AGAIN-1, E28-AGAIN-2, E37-2, E38-1, E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:
A small-size agarose gel was prepared for gel electrophoresis.
Unfortunately insert length was correct only for E28-AGAIN-1 so we ligated again E41:
Glycerol stocks were prepared according to the protocol for J101-31, J101-E5 and J101-E7.
August, 3rd
T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 μl of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
Then samples were centrifuged at 13.000 rpm; finally the supernatant was collected and stored at -20°C.
|
Team:UNIPV-Pavia/Calendar/August/week1
From 2011.igem.org
(Difference between revisions)
Line 144: | Line 144: | ||
<td>16.5</td> | <td>16.5</td> | ||
<td>0.5 EcoRI</td> | <td>0.5 EcoRI</td> | ||
- | <td>0.5 | + | <td>0.5 PstI</td> |
<td>2.5</td> | <td>2.5</td> | ||
<td>25</td> | <td>25</td> | ||
Line 161: | Line 161: | ||
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_02_08_2011_E41-AGAIN-1_EP_E41-AGAIN-2_EP_E28-AGAIN-1_EP_E28-AGAIN-2_EP.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/6f/UNIPV_02_08_2011_E41-AGAIN-1_EP_E41-AGAIN-2_EP_E28-AGAIN-1_EP_E28-AGAIN-2_EP.png" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Smallsize gel</div></div></div></div> | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_02_08_2011_E41-AGAIN-1_EP_E41-AGAIN-2_EP_E28-AGAIN-1_EP_E28-AGAIN-2_EP.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/6f/UNIPV_02_08_2011_E41-AGAIN-1_EP_E41-AGAIN-2_EP_E28-AGAIN-1_EP_E28-AGAIN-2_EP.png" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Smallsize gel</div></div></div></div> | ||
- | |||
<p> | <p> |
Revision as of 21:15, 16 August 2011