Team:Calgary/27 July 2009

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Revision as of 16:03, 29 July 2009

University of Calgary

THIS MONTH

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NOTEBOOK PAGE INDEX

Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki

CALENDAR

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JULY 27, 2009

CAROL

Finishing Modeling Paper

Finished writing modeling paper that summarizes what we will be doing once the circuit is completed. The experiments should not take long to accomplished and these results can be tabulated in Matlab quickly.
Plated the week-long ligation of the promoter library in both XL Gold Ultra competent cells and Top 10 cells, but there is no growth.
A reason that we thought of that might have caused minimal colonies is the phosphatase step. The 32 primers that we have synthesized does not have 5' phosphates. If we phosphatase the vector, that might result in no ligation.
Re-started restriction enzyme digestion on vector pCS26 with XhoI and BamHI at 37^oC for 4 hours. Then I ligated the promoter with the vector with Quick Ligase for 5 minutes. Then I transformed the plasmids into both XL Gold Ultracompetent cells and Top 10 cells. Incubate at 37^oC for 16 hours.

CHINMOYEE

Descriptive Title of What You're Doing

WIKI CODING HERE

EMILY

Descriptive Title of What You're Doing

WIKI CODING HERE

FAHD

Descriptive Title of What You're Doing

WIKI CODING HERE

IMAN

Descriptive Title of What You're Doing

WIKI CODING HERE

JAMIE

Descriptive Title of What You're Doing

WIKI CODING HERE

JEREMY

Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers

Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.

Protocol

	MM1 (6x) (μL)	MM2 (6x) (μL)
10X PCR buffer minus MgCl₂	30	30
10mM dNTPs	6	6
50mM MgCl₂	9	9
F primer	6 (BBK CP F)	6 (LuxPQ F)
R primer	6 (BBK CP R)	6 (LuxOU R)
ddH₂O	241.8	241.8
pTaq	1.2	1.2

Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3

PCR conditions

Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.

Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers

Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)

Protocol

Positive control = ∆LuxPQ in psB1AK3

PCR conditions

Result: Of the 6 colonies, 5 had bands of the correct size (~3.9kb).

Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)

Purpose: isolate and measure concentrations of pure plasmids.

Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Protocol

Recipient

Insert

Put in the incubator at 37ºC overnight.

KATIE

Descriptive Title of What You're Doing

WIKI CODING HERE

KEVIN

Descriptive of What You're Doing

WIKI CODING HERE

MANDY

Descriptive Title of What You're Doing

WIKI CODING HERE

PATRICK

Descriptive Title of What You're Doing

WIKI CODING HERE

PRIMA

Descriptive Title of What You're Doing

WIKI CODING HERE

STEFAN

Descriptive Title of What You're Doing

WIKI CODING HERE

VICKI

Descriptive Title of What You're Doing

WIKI CODING HERE

@@ Line 320: / Line 320: @@
 <div class="desc">
 </html>
-Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.
+<html>
+Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers
+Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.
+<br>
+<br>
 Protocol
-	MM1 (6x) (μL)	MM2 (6x) (μL)
+<html>
-X PCR buffer minus MgCl2	30	30
+<table x:str border=0 cellpadding=0 cellspacing=0 width=404 style='border-collapse:
-mM dNTPs	6	6
+ collapse;table-layout:fixed;width:304pt'>
-mM MgCl2	9	9
+ <col width=194 style='mso-width-source:userset;mso-width-alt:7094;width:146pt'>
-F primer	6 (BBK CP F)	6 (LuxPQ F)
+ <col width=105 span=2 style='mso-width-source:userset;mso-width-alt:3840;
-R primer	6 (BBK CP R)	6 (LuxOU R)
+ width:79pt'>
-ddH2O	241.8	241.8
+ <tr height=22 style='height:16.5pt;mso-yfti-firstrow:yes;mso-yfti-irow:0'>
-pTaq	1.2	1.2
+  <td height=22 class=xl24 width=194 style='height:16.5pt;width:146pt'>&nbsp;</td>
+  <td class=xl25 width=105 style='width:79pt'>MM1 (6x) (&#956;L)</td>
+  <td class=xl25 width=105 style='width:79pt'>MM2 (6x) (&#956;L)</td>
+ </tr>
+ <tr height=26 style='height:19.5pt'>
+  <td height=26 class=xl26 width=194 style='height:19.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>10X PCR buffer minus MgCl<font
+  class="font6"><sub>2</sub></font></span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>30</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>30</span></td>
+ </tr>
+ <tr height=22 style='height:16.5pt'>
+  <td height=22 class=xl26 width=194 style='height:16.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>10mM dNTPs</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>6</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>6</span></td>
+ </tr>
+ <tr height=26 style='height:19.5pt'>
+  <td height=26 class=xl26 width=194 style='height:19.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>50mM MgCl<font class="font6"><sub>2</sub></font></span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>9</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>9</span></td>
+ </tr>
+ <tr height=22 style='height:16.5pt'>
+  <td height=22 class=xl26 width=194 style='height:16.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>F primer</span></td>
+  <td class=xl27 width=105 style='width:79pt'><span lang=DA style='mso-ansi-language:
+  DA'>6 (BBK CP F)</span></td>
+  <td class=xl27 width=105 style='width:79pt'><span lang=DA style='mso-ansi-language:
+  DA'>6 (LuxPQ F)</span></td>
+ </tr>
+ <tr height=22 style='height:16.5pt'>
+  <td height=22 class=xl26 width=194 style='height:16.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>R primer</span></td>
+  <td class=xl27 width=105 style='width:79pt'><span lang=DA style='mso-ansi-language:
+  DA'>6 (BBK CP R)</span></td>
+  <td class=xl27 width=105 style='width:79pt'><span lang=DA style='mso-ansi-language:
+  DA'>6 (LuxOU R)</span></td>
+ </tr>
+ <tr height=26 style='height:19.5pt'>
+  <td height=26 class=xl26 width=194 style='height:19.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>ddH<font class="font6"><sub>2</sub></font><font
+  class="font5">O</font></span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>241.8</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>241.8</span></td>
+ </tr>
+ <tr height=22 style='height:16.5pt'>
+  <td height=22 class=xl26 width=194 style='height:16.5pt;width:146pt'><span
+  lang=DA style='mso-ansi-language:DA'>pTaq</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>1.2</span></td>
+  <td class=xl27 width=105 style='width:79pt' x:num><span lang=DA
+  style='mso-ansi-language:DA'>1.2</span></td>
+ </tr>
+ <![if supportMisalignedColumns]>
+ <tr height=0 style='display:none'>
+  <td width=194 style='width:146pt'></td>
+  <td width=105 style='width:79pt'></td>
+  <td width=105 style='width:79pt'></td>
+ </tr>
+ <![endif]>
+</table>
+</html>
 Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3
 PCR conditions
-# of cycles	Temp (ºC)	Time
-	94	6 min
-	94	30sec
-	45sec
-	6min 20sec
-	72	10min
-	hold
 Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.
@@ Line 367: / Line 443: @@
 Protocol
-	MM1 (8x) (μL)	MM2 (6x) (μL)
-X PCR buffer minus MgCl2	40	40
-mM dNTPs	8	8
-mM MgCl2	12	12
-F primer	8 (LuxPQ F)	8 (BBK CP F)
-R primer	8 (LuxPQ R)	8 (BBK CP R)
-ddH2O	322.4	322.4
-pTaq	1.6	1.6
 Positive control = ∆LuxPQ in psB1AK3
 PCR conditions
-# of cycles	Temp (ºC)	Time
-	94	6 min
-	94	30sec
-	45sec
-	6min 20sec
-	72	10min
-	hold
@@ Line 397: / Line 459: @@
 Purpose: isolate and measure concentrations of pure plasmids.
-DNA	260/280	260/230	Conc. [ng/μL]
-∆LuxPQ in AC3 C1	1.83	2.23	146.8
-∆LuxPQ in AC3 C2	1.81	2.19	112.6
-∆LuxPQ in AC3 C3	1.80	2.27	145.8
-∆LuxPQ in AC3 C4	1.78	2.09	96.6
-∆LuxPQ in AC3 C5	1.81	2.25	115.3
-∆LuxPQ in AC3 C6	1.79	2.17	44.8
-PQ-B-R-OU-B in AC3 C1	1.76	1.86	29.4
-PQ-B-R-OU-B in AC3 C2	1.59	1.44	20.1
-PQ-B-R-OU-B in AC3 C3	1.70	2.27	16.3
-PQ-B-R-OU-B in AC3 C4	1.71	1.79	17.0
@@ Line 425: / Line 477: @@
 Recipient
-Recipient 1	Recipient 2
-μL REact 4	2μL REact 4
-.75 μL SpeI	0.75 μL SpeI
-.75 μL PstI	0.75 μL PstI
-μL of ∆LuxPQ in AK3 C2 [112.6ng/μL]	2 μL of ∆LuxPQ in AK3 C5 [115.3ng/μL]
-.5 μL ddH2O	14.5 μL ddH2O
 Insert
-μL REact 2
-.75 μL XbaI
-.75 μL PstI
-μL of LuxPQ-B0015-R0040-LuxOU-B0015 in AK3 C1 [156.3ng/μL]
-.5 μL ddH2O
 Put in the incubator at 37ºC overnight.