Team:Goettingen/week21-2

From 2012.igem.org

(Difference between revisions)

Revision as of 08:33, 21 September 2012

Language:

English,

Deutsch

#2 Speed Improvement - 21st week

Back to overview

V09_17

V09_17_1 Preparative double digestion of flhDC, FliC, 18K-RFP and pSB1C3 followed by ligation

Experiment:
In order to clone the flhDC, FliC and 18K-RFP constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.

V09_17_2 Preparation of over night cultures

Experiment: We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used:

- 20E_flhDC
- 18C_flhDC
- 20I_flhdc
- 20E_motA
- 18C_motA
- 20E_motB
- 18C_motB

V09_18

V09_18_1 Motility Assays

Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
Observation and Results:
Text

V09_18_2 Preparative double digestion of MotA, MotB, yhjH, 18C-RFP, 20E-RFP and 20I-RFP followed by ligation

Experiment:
TEXT

V09_18_3 Chemical transformation of different plasmid-gene constructs into E. coli (DH10B) or (MG1655)

Experiment:
Chemical transformation was done according to the standard protocol. The following constructs were used:
- pSB1C3-FliC into E. coli (DH10B)
pSB1C3-flhDC into E. coli (DH10B)
PSB1C3-RFP into E. coli (MG1655)

V09_19

V09_19_1 Motility Assays

Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
Observation and Results:
Text

V09_19_2 Chemical transformation

Experiment:
For the chemical retransformation the standard protocol was followed. The following constructs were transformed:
- pSB1C3-18C-motA (DH10B)
- pSB1C3-18C-motB (DH10B)
- pSB1C3-18C-yhjH (DH10B)
- pSB1C3-20E-motA (DH10B)
- pSB1C3-20E-motA (DH10B)
- pSB1C3-20E-motA (DH10B)
- pSB1C3-20I-motA (DH10B)
- pSB1C3-20I-motB (DH10B)
- pSB1C3-20I-yhjH (DH10B)
- pSB1C3-RFP (BL21)

V09_19_3 Preparation of over night cultures

Experiment:
Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
- pSB1C3-flhDC
- pSB1C3-fliC
- pSB1C3-18K-RFP

V09_20

V09_20_1 Mini Prep and test digestion of pSB1C3-gene constructs

Experiment:
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.
Observation and Results:
All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.

V09_20_2 PCR of fliC

Experiment:
In order to get more fliC, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.

V09_20_3 Repetition of preparative double digestion of flhDC, FliC and pSB1C3

Experiment:
In order to clone the flhDC, FliC constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol.

↑ Return to top

Important pages:
Home; Team; Official Team Profile; Project; Parts submitted to the Registry; Modeling; Notebook; Saftey; Attributions

@@ Line 616: / Line 616: @@
 - pSB1C3-<i>fliC</i> <br>
 - pSB1C3-18K-<i>RFP</i> <br>
+<br>
+<br></td></tr>
+</table>
+<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
+<tr bordercolor="black" valign="top">
+<td width="900" bordercolor="black" valign="top">
+<h2><b>V09_20 </b></h2><br>
+<b> V09_20_1 Mini Prep and test digestion of pSB1C3-gene constructs</i></i></b><br>
+<ul>
+<li>Experiment: <br>
+Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol. </a> <br>
+<li> Observation and Results:<br>
+All three the pSB1C3-18K-<i>RFP</i> clones host the correctly inserted gene in the plasmid. The pSB1C3-<i>flhDC / fliC</i> clones do not host the correctly inserted gene.<br>
+</ul>
+<br>
+<b>V09_20_2 PCR of <i>fliC</i></b><br>
+<ul>
+<li>Experiment: <br>
+In order to get more <i>fliC</i>, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.<br>
+</ul>
+<br>
+<b>V09_20_3 Repetition of preparative double digestion of <i>flhDC, FliC</i> and <i> pSB1C3</i></b><br>
+<ul>
+<li>Experiment:<br> In order to clone the flhDC, FliC constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol.
 <br>
 <br></td></tr>