Team:Paris Saclay/Notebook/July/4

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=='''Lab work'''==
=='''Lab work'''==
-
constructing
+
*'''A.aero/anaerobic regulation system'''
 +
**''2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3 ''
 +
**''BioBrick RBS+amilCP+terminator in plasmid PSB1C3 ''
 +
 
 +
<u>Electrophoresis band size estimation</u>
 +
<p>We used Clonemanager for band size estimation:</p>
 +
<br>
 +
{| border="1" align="center"
 +
|-
 +
|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
 +
|}<br>
 +
 
 +
 
 +
<u>PCR interpretation</u><br>
 +
 
 +
{| align="center"
 +
| style="width:350px;border:1px solid black;" | IMAGE
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
*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%
 +
|}
 +
 
 +
*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
 +
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>
 +
<u>Stock BBa_K1155000</u><br>
 +
1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.
 +
<u>Plasmid DNA extraction</u><br>
 +
From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.<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.
 +
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
 +
 
 +
 

Revision as of 23:10, 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

IMAGE
  • 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%
  • 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

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




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