Team:Paris Saclay/Notebook/July/4

From 2013.igem.org

(Difference between revisions)
(→Lab work)
(→Lab work)
Line 45: Line 45:
{| align="center"
{| align="center"
-
| style="width:350px;border:1px solid black;" | IMAGE
+
| style="width:350px;border:1px solid black;" | [[File:PCRPS040713.jpg|right|350px]]
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
+
| style="width:350px;border:1px solid black;" |
*Well 1,2,5,6,10,11 : plasmid without fnr
*Well 1,2,5,6,10,11 : plasmid without fnr
*Well 3,4,8,9,12,13 : plasmid with fnr
*Well 3,4,8,9,12,13 : plasmid with fnr
Line 53: Line 53:
*Well 10 to 13 : primer fnr_up/vr
*Well 10 to 13 : primer fnr_up/vr
*gel 1.5%
*gel 1.5%
-
|}
+
|}<br>
-
*Well 1,2,5,6,10,11 : plasmid without fnr
+
<p>We observed for well 10 and well 11 there were a problem which could be some fault in the manipulation. However, the well 4,9,13 conformed to our estimation.</p>
-
*Well 3,4,8,9,12,13 : plasmid with fnr
+
-
*Well 1 to 4 : primer vf/vr
+
-
*Well 5,6,,8,9 : primer vf/fnr_down
+
-
*Well 10 to 13 : primer fnr_up/vr
+
-
We observed for well 10 and well 11 there were a problem which could be some fault in the manipulation. However, the well 4,9,13 conformed to our estimation.
+
<br>
<br>
<u>Stock BBa_K1155000</u><br>
<u>Stock BBa_K1155000</u><br>
-
1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.
+
<p>1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.</p><br>
<u>Plasmid DNA extraction</u><br>
<u>Plasmid DNA extraction</u><br>
From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.<br>
From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.<br>
<u>Restriction digest</u><br>
<u>Restriction digest</u><br>
-
We used 3 enzymes of restriction, they were Not I, Mlu I and Hpa I. And we prepared 2 * 3 = 6 tubes.
+
<p>We used 3 enzymes of restriction, they were Not I, Mlu I and Hpa I. And we prepared 2 * 3 = 6 tubes.
-
So we add into each tube:
+
So we add into each tube:</p>
-
Extracted DNA solution : 2µl
+
*Extracted DNA solution : 2µl
-
Buffer Oranger : 2µl
+
*Buffer Oranger : 2µl
-
Enzyme : 0.5µl
+
*Enzyme : 0.5µl
-
H2O : 15.5µl
+
*H2O : 15.5µl
-
Total : 20µl
+
*Total : 20µl
-
The incubation was at 37°C during 90min
+
<p>The incubation was at 37°C during 90min</p>

Revision as of 23:15, 18 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Notebook : July 4

Summary:

For regulator system:
  • estimated the size of segments for bands of electrophoresis by using Clone Manager.
  • Made interpretation for electrophoresis. The picture shows that the experimental result was coherent with our estimation. We constructed our first BrioBrick, BBa_K1155000 (fnr+plasmid PSB1C3).
  • stored 2 colonies who contain BioBrick BBa_K1155000
  • the plasmid DNA extraction was performed for BBa_K1155000.
  • The extract of plasmid DNA which contain BBa_K1155000 was digested by Not I, Mlu I, Hpa I, and were amplified by PCR.
  • Seeded the 4 additional broths (2 for amilCP, 2 for FNR) for plasmid extraction(mini prep).

For PCBs sensor system:

  • Received the bacterial strain: pseudomonas KE707.

Lab work

  • A.aero/anaerobic regulation system
    • 2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
    • BioBrick RBS+amilCP+terminator in plasmid PSB1C3

Electrophoresis band size estimation

We used Clonemanager for band size estimation:


molecule Primer pair Size
Plasmid without fnr VF/VR 277bp
Plasmid with fnr Pfnr_up/Pfnr_down 276bp
Plasmid with fnr Pfnr_up/VR 311bp


PCR interpretation

PCRPS040713.jpg
  • Well 1,2,5,6,10,11 : plasmid without fnr
  • Well 3,4,8,9,12,13 : plasmid with fnr
  • Well 1 to 4 : primer vf/vr
  • Well 5,6,,8,9 : primer vf/fnr_down
  • Well 10 to 13 : primer fnr_up/vr
  • gel 1.5%

We observed for well 10 and well 11 there were a problem which could be some fault in the manipulation. However, the well 4,9,13 conformed to our estimation.


Stock BBa_K1155000

1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.


Plasmid DNA extraction
From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.
Restriction digest

We used 3 enzymes of restriction, they were Not I, Mlu I and Hpa I. And we prepared 2 * 3 = 6 tubes. So we add into each tube:

  • Extracted DNA solution : 2µl
  • Buffer Oranger : 2µl
  • Enzyme : 0.5µl
  • H2O : 15.5µl
  • Total : 20µl

The incubation was at 37°C during 90min




Previous day Back to calendar Next day

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/July/4"