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Revision as of 09:42, 1 October 2010 (view source) Louch07 (Talk | contribs) (→Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers) ← Older edit		Latest revision as of 19:21, 16 October 2010 (view source) Toand (Talk | contribs) (→Lab notes (8/30 - 9/5))
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-	=== Two-step PCR of miniprep ===	+	== Photosensor ==
-	<br>	+	=== PCR on pKJ606 with PSfw and VR primers ===
-	==== First step: PCR of miniprep with mutation primers ====	+	Date: 31/8<br>https://2010.igem.org/wiki/index.php?title=Team:SDU-Denmark/labnotes8&action=edit&section=3
-	<br>	+	Done by: LC<br>
-	''Done by:'' Louise <br>	+	Methods: PCR<br>
-	''Date:'' September 7th <br>	+	Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
-	''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>	+	Notes: <br>
-	''Notes:'' <br>	+	Premix:<br>
-	'''3 x Premix 1:''' <br>	+	7,5 µl 10xTAQ Buffer<br>
-	114.3ul water <br>	+	3 µl MgCl2 <br>
-	15ul Pfu Buffer <br>	+	3 µl VF2 <br>
-	4.5ul dNTP <br>	+	3 µl VR <br>
-	3ul MgSO4 <br>	+	1,5 µl dNTP<br>
-	4.5ul FlhDC fw <br>	+	55,5 µl H2O<br>
-	4.5ul FlhDCmut rev <br>	+	3 µl template (PS1 miniprep)<br>
-	1.2ul PFU <br>	+	3/8 µl TAQ Polymerase<br>
-	3ul template (miniprep) <br><br>	+
-	'''3 x Premix 2:''' <br>	+
-	114.3ul water <br>	+
-	15ul Pfu Buffer <br>	+
-	4.5ul dNTP <br>	+
-	3ul MgSO4 <br>	+
-	4.5ul FlhDCmut fw <br>	+
-	4.5ul FlhDC rev <br>	+
-	1.2ul PFU <br>	+
-	3ul template (miniprep) <br><br>	+
-	''Results:''	+
-	<br>	+
-	NanoDrop: <br>	+
-	'''Sample 1.1:''' Concentration: 359.5ng/ul Purity: 1.83/2.20 <br>	+
-	'''Sample 2.1:''' Concentration: 304.1ng/ul Purity: 1.85/2.23 <br>	+
-	<br>	+
-	The PCR samples were run on a 1.5% gel with a 100bp-1000bp ladder. The first three samples are all positioned between 100bp and 200bp, this is the part of the gene run with FlhDCmut fv and FlhDC rev primers. The other three samples are all positioned betveen 900bp and 1000bp, this is the pcr with FlhDC fv and FlhDCmut rev primers. All samples looked okay and were pooled as sample 1.1 (light band) and sample 2.1 (heavy band)<br>	+
-	[[Image:Team SDU-Denmark-Pfx pcr af miniprep.jpg|300px]]	+
-	<br>	+
-	A gel extraction of the PCR product of sample 1.1 and 2.1 were made:	+
	<br>		<br>
-	[[Image:Team SDU-Denmark Gel extraction af FlhDCmut dele.jpg|300px]]	+	PCR Program:<br>
-	<br>	+	<table style="text-align: left;" border="1" cellpadding="2"
-	The gel extraction products were used in the preceding PCRs.	+	cellspacing="2">
-		+
-	==== Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers ====	+
-	<br>	+
-	''Done by:'' Maria <br>	+
-	''Date:'' September 9th <br>	+
-	''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>	+
-	''Notes:'' <br>	+
-	'''Premix:''' <br>	+
-	38ul water <br>	+
-	5ul PFU buffer + MgSO4 <br>	+
-	1.5ul dNTP <br>	+
-	1ul Sample 1.1 <br>	+
-	1ul Sample 2.1 <br>	+
-	1.5ul FlhDC fw primer <br>	+
-	1.5ul FlhDC rev primer <br>	+
-	0.5ul PFU <br><br>	+
-	'''PCR Program:''' <br>	+
-	<table style="text-align: left; height: 260px; width: 225px;" border="1"	+
-	cellpadding="2" cellspacing="2">	+
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">1:Start<br>	+	<td style="vertical-align: top;">Start<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">95C<br>	+	<td style="vertical-align: top;">94&nbsp; C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 min<br>	+	<td style="vertical-align: top;">2 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">2: Denaturing<br>	+	<td style="vertical-align: top;">Denaturing<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">95C<br>	+	<td style="vertical-align: top;">94 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">30 sec<br>	+	<td style="vertical-align: top;">1 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">3: Annealing<br>	+	<td style="vertical-align: top;">Annealing<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">56C<br>	+	<td style="vertical-align: top;">55 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">30 sec<br>	+	<td style="vertical-align: top;">1 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">4: Elongation<br>	+	<td style="vertical-align: top;">Elongation<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">72C<br>	+	<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 min<br>	+	<td style="vertical-align: top;">3 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">5:<br>	+	<td style="vertical-align: top;">Goto2<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">GO TO<br>	+	<td style="vertical-align: top;">rep<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 rep. 4x<br>	+	<td style="vertical-align: top;">29x<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">6: Denaturing<br>	+	<td style="vertical-align: top;">End<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">95C<br>	+	<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">30 sec<br>	+	<td style="vertical-align: top;">3 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">7: Annealing<br>	+	<td style="vertical-align: top;">Hold<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">63C<br>	+	<td style="vertical-align: top;">4 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">30 sec<br>	+	<td style="vertical-align: top;"><br>
	</td>		</td>
	</tr>		</tr>
		+	</table>
		+	<br>
		+	<br>
		+	Results: The bands that showed up were around 2500 BP as expected from the sequencing results. This means that the designed primers have a high possibility of working, so that they will be ordered.<br>
		+	[[Image:Team-SDU-Denmark-PSfwVR.jpg|400px]]
		+	<br>
		+
		+	=== PCR on pKJ606 with PSfw and PSrv primers ===
		+	Date: 02/09<br>
		+	Done by: LC<br>
		+	Methods: PCR<br>
		+	Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
		+	Notes: <br>
		+	Premix:<br>
		+	7,5 µl 10xTAQ Buffer<br>
		+	3 µl MgCl2 <br>
		+	3 µl VF2 <br>
		+	3 µl VR <br>
		+	1,5 µl dNTP<br>
		+	55,5 µl H2O<br>
		+	3 µl template (PS1 miniprep)<br>
		+	3/8 µl TAQ Polymerase<br>
		+	<br>
		+	PCR Program:<br>
		+	<table style="text-align: left;" border="1" cellpadding="2"
		+	cellspacing="2">
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">8: Elongation<br>	+	<td style="vertical-align: top;">Start<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">72C<br>	+	<td style="vertical-align: top;">94&nbsp; C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 min<br>	+	<td style="vertical-align: top;">2 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">9:<br>	+	<td style="vertical-align: top;">Denaturing<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">GO TO<br>	+	<td style="vertical-align: top;">94 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">6 rep. 25x<br>	+	<td style="vertical-align: top;">1 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">10: End <br>	+	<td style="vertical-align: top;">Annealing<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">72C<br>	+	<td style="vertical-align: top;">55 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">5 min<br>	+	<td style="vertical-align: top;">1 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">12: Hold<br>	+	<td style="vertical-align: top;">Elongation<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">4C<br>	+	<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;"><br>	+	<td style="vertical-align: top;">2 min<br>
	</td>		</td>
	</tr>		</tr>
-	</table> <br><br>	+	<tr>
-		+	<td style="vertical-align: top;">Goto2<br>
-	''Results:'' <br>	+	</td>
-	Some of the PCR product was run on a 1.5% gel with a 10kb ladder. <br>	+	<td style="vertical-align: top;">rep<br>
-	[[Image:Team SDU-Denmark God pcr af FlhDCmut (maria) 1.jpg|300px]]	+	</td>
		+	<td style="vertical-align: top;">29x<br>
		+	</td>
		+	</tr>
		+	<tr>
		+	<td style="vertical-align: top;">End<br>
		+	</td>
		+	<td style="vertical-align: top;">72 C<br>
		+	</td>
		+	<td style="vertical-align: top;">3 min<br>
		+	</td>
		+	</tr>
		+	<tr>
		+	<td style="vertical-align: top;">Hold<br>
		+	</td>
		+	<td style="vertical-align: top;">4 C<br>
		+	</td>
		+	<td style="vertical-align: top;"><br>
		+	</td>
		+	</tr>
		+	</table>
	<br>		<br>
-	The rest of the product was extracted from a new gel, unfortunately there were problems with the camera, so there is no picture of the gel extraction.
	<br>		<br>
-	===== PCR of Gel extraction =====	+	Results: The bands that showed up were around 1750 BP, further confirming sequencing results. New primers for taking the whole gene out have been ordered.<br>
		+	[[Image:Team-SDU-Denmark-PSfwrv.jpg|400px]]
	<br>		<br>
-	''Done by:'' Maria <br>
-	''Date:'' September 9th <br>
-	''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
-	''Notes:'' <br>
-	'''Premix x 6:''' <br>
-	234ul water <br>
-	30ul PFU buffer + MgSO4 <br>
-	9ul dNTP <br>
-	9ul FlhDC fw primer <br>
-	9ul FlhDC rev primer <br>
-	2.5ul PFU <br>
-	6ul template <br>
-	<table style="text-align: left; height: 260px; width: 225px;" border="1"	+	=== PCR on pKJ606 with fwPS2 and rvPS2 primers ===
-	cellpadding="2" cellspacing="2">	+	Date: 04/09<br>
		+	Done by: LC<br>
		+	Methods: PCR<br>
		+	Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
		+	Notes: <br>
		+	Premix:<br>
		+	12,5 µl 10xTAQ Buffer<br>
		+	5 µl MgCl2 <br>
		+	5 µl VF2 <br>
		+	5 µl VR <br>
		+	2,5 µl dNTP<br>
		+	59,5 µl H2O<br>
		+	4 µl template (Consisting of 2 µl H2O and 2 µl of pKJ606 miniprep)<br>
		+	1/2 µl TAQ Polymerase<br>
		+	<br>
		+	PCR Program:<br>
		+	<table style="text-align: left;" border="1" cellpadding="2"
		+	cellspacing="2">
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">1:Start<br>	+	<td style="vertical-align: top;">Start<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">95C<br>	+	<td style="vertical-align: top;">94&nbsp; C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 min<br>	+	<td style="vertical-align: top;">2 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">2: Denaturing<br>	+	<td style="vertical-align: top;">Denaturing<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">95C<br>	+	<td style="vertical-align: top;">94 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">30sec<br>	+	<td style="vertical-align: top;">1 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">3: Annealing<br>	+	<td style="vertical-align: top;">Annealing<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">63C<br>	+	<td style="vertical-align: top;">55 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">30 sec<br>	+	<td style="vertical-align: top;">1 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">4: Elongation<br>	+	<td style="vertical-align: top;">Elongation<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">72C<br>	+	<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 min<br>	+	<td style="vertical-align: top;">2:30 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">5:<br>	+	<td style="vertical-align: top;">Goto2<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">GO TO<br>	+	<td style="vertical-align: top;">rep<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">2 rep. 29x<br>	+	<td style="vertical-align: top;">29x<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">6: End <br>	+	<td style="vertical-align: top;">End<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">72C<br>	+	<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;">5 min<br>	+	<td style="vertical-align: top;">3 min<br>
	</td>		</td>
	</tr>		</tr>
	<tr>		<tr>
-	<td style="vertical-align: top; width: 102px;">7: Hold<br>	+	<td style="vertical-align: top;">Hold<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 52px;">4C<br>	+	<td style="vertical-align: top;">4 C<br>
	</td>		</td>
-	<td style="vertical-align: top; width: 45px;"><br>	+	<td style="vertical-align: top;"><br>
	</td>		</td>
	</tr>		</tr>
-	</table> <br><br>	+	</table>
	<br>		<br>
	<br>		<br>
-	''Results:''	+	Results: Primers were confirmed working, the next step will be PFU pcr.<br>
-	<br>	+
-	The PCR worked fine, unfortunately there were problems with the camera, so there is no picture of the gel.	+
-	== Photosensor ==	+	== Retinal ==
-	=== PCR on pKJ606 with PSfw and VR primers ===	+
-	Date: 31/8<br>https://2010.igem.org/wiki/index.php?title=Team:SDU-Denmark/labnotes8&action=edit&section=3	+	=== Gradient PCR on ninaB (after PCR with ninaB2fw and ninaB2rv) with ninaBfw and ninaBrv ===
-	Done by: LC<br>	+	Date: 30/08<br>
		+	Done by: Tommy & Marie<br>
	Methods: PCR<br>		Methods: PCR<br>
-	Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>	+	Protocols: CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]<br>
	Notes: <br>		Notes: <br>
-	Premix:<br>	+	To finde the optimal anneling temperatur a gradient PCR from 60 to 75 degrees<br>
-	7,5 µl 10xTAQ Buffer<br>	+	"ninaB" was used as template and ninaB2fw and ninaB2rv was used as primers<br>
-	3 µl MgCl2 <br>	+
-	3 µl VF2 <br>	+
-	3 µl VR <br>	+
-	1,5 µl dNTP<br>	+
-	55,5 µl H2O<br>	+
-	3 µl template (PS1 miniprep)<br>	+
-	3/8 µl TAQ Polymerase<br>	+
-	<br>	+
	PCR Program:<br>		PCR Program:<br>
	<table style="text-align: left;" border="1" cellpadding="2"		<table style="text-align: left;" border="1" cellpadding="2"
Line 300:		Line 304:
	<td style="vertical-align: top;">Annealing<br>		<td style="vertical-align: top;">Annealing<br>
	</td>		</td>
-	<td style="vertical-align: top;">55 C<br>	+	<td style="vertical-align: top;">Grad. C<br>
	</td>		</td>
	<td style="vertical-align: top;">1 min<br>		<td style="vertical-align: top;">1 min<br>
Line 310:		Line 314:
	<td style="vertical-align: top;">72 C<br>		<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top;">3 min<br>	+	<td style="vertical-align: top;">4 min<br>
	</td>		</td>
	</tr>		</tr>
Line 326:		Line 330:
	<td style="vertical-align: top;">72 C<br>		<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top;">3 min<br>	+	<td style="vertical-align: top;">5 min<br>
	</td>		</td>
	</tr>		</tr>
Line 338:		Line 342:
	</tr>		</tr>
	</table>		</table>
-	<br>	+	Vary small amount of the product are observed, it is pooled and gel purifid, the nanodrop shows 372,3 and 377,1 ng/uL.
-	<br>	+
-	Results: The bands that showed up were around 2500 BP as expected from the sequencing results. This means that the designed primers have a high possibility of working, so that they will be ordered.<br>	+
-	[[Image:Team-SDU-Denmark-PSfwVR.jpg|400px]]	+
-	<br>	+
-	=== PCR on pKJ606 with PSfw and PSrv primers ===	+	=== Gel purification af ninaB from gradient PCR ===
-	Date: 02/09<br>	+	Date: 31/08<br>
-	Done by: LC<br>	+	Done by: Tommy & Marie<br>
		+	Methods: PCR<br>
		+	Protocols: CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]<br>
		+	Notes: <br>
		+	Gel purification was preformed on the pooled product from the gradient PCR according to the protocol<br>
		+	After the gel purification the 2 samples was pooled and nanodroped: 7.0 ng/ul<br>
		+	Furthere PCR was preformed on the purifid product<br>
		+
		+
		+	=== Gel purification af ninaB from gradient PCR ===
		+	Date: 1/09<br>
		+	Done by: Tommy & Marie<br>
	Methods: PCR<br>		Methods: PCR<br>
	Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>		Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
	Notes: <br>		Notes: <br>
-	Premix:<br>	+	PCR was run according to protocol to the following to this program<br>
-	7,5 µl 10xTAQ Buffer<br>	+	Gel purified produckt was used as template and ninaBfw and ninaBrv was used as primers<br>
-	3 µl MgCl2 <br>	+
-	3 µl VF2 <br>	+
-	3 µl VR <br>	+
-	1,5 µl dNTP<br>	+
-	55,5 µl H2O<br>	+
-	3 µl template (PS1 miniprep)<br>	+
-	3/8 µl TAQ Polymerase<br>	+
-	<br>	+
	PCR Program:<br>		PCR Program:<br>
	<table style="text-align: left;" border="1" cellpadding="2"		<table style="text-align: left;" border="1" cellpadding="2"
Line 382:		Line 385:
	<td style="vertical-align: top;">Annealing<br>		<td style="vertical-align: top;">Annealing<br>
	</td>		</td>
-	<td style="vertical-align: top;">55 C<br>	+	<td style="vertical-align: top;">Grad. C<br>
	</td>		</td>
	<td style="vertical-align: top;">1 min<br>		<td style="vertical-align: top;">1 min<br>
Line 392:		Line 395:
	<td style="vertical-align: top;">72 C<br>		<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top;">2 min<br>	+	<td style="vertical-align: top;">4 min<br>
	</td>		</td>
	</tr>		</tr>
Line 400:		Line 403:
	<td style="vertical-align: top;">rep<br>		<td style="vertical-align: top;">rep<br>
	</td>		</td>
-	<td style="vertical-align: top;">29x<br>	+	<td style="vertical-align: top;">44x<br>
	</td>		</td>
	</tr>		</tr>
Line 408:		Line 411:
	<td style="vertical-align: top;">72 C<br>		<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top;">3 min<br>	+	<td style="vertical-align: top;">5 min<br>
	</td>		</td>
	</tr>		</tr>
Line 420:		Line 423:
	</tr>		</tr>
	</table>		</table>
-	<br>
-	<br>
-	Results: The bands that showed up were around 1750 BP, further confirming sequencing results. New primers for taking the whole gene out have been ordered.<br>
-	[[Image:Team-SDU-Denmark-PSfwrv.jpg|400px]]
-	<br>
-	=== PCR on pKJ606 with fwPS2 and rvPS2 primers ===	+	=== Restriction digest of ninaB ===
-	Date: 04/09<br>	+	Date: 2/09<br>
-	Done by: LC<br>	+	Done by: Tommy & Marie<br>
		+	Methods: restriction digest<br>
		+	Protocols: RD1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1]<br>
		+	Notes: <br>
		+	EcoRI and PstI ristriction enzymes was used and the ristriction digest was preformed according to protocol<br>
		+	No gel purification was preformed, purificatin was preformed directly from the restriction mixture<br>
		+	Purification was deno using the GFX purification kit and proformed according to that protocol, the purifid product was nanodroped: 3,1 ng/uL
		+
		+	=== "Cross-PCR" on ninaB ===
		+	Date: 2/09<br>
		+	Done by: Tommy & Marie<br>
	Methods: PCR<br>		Methods: PCR<br>
-	Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>	+	Protocols: CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]<br>
	Notes: <br>		Notes: <br>
-	Premix:<br>	+	Tubes A: ninaBfw and ninaB2rv<br>
-	12,5 µl 10xTAQ Buffer<br>	+	Tubes B: ninaB2fw and ninaBrv<br>
-	5 µl MgCl2 <br>	+	NinaB PCR product with ninaB2fw and rv was used as template, both the tubes was run on the same PCR program
-	5 µl VF2 <br>	+
-	5 µl VR <br>	+
-	2,5 µl dNTP<br>	+
-	59,5 µl H2O<br>	+
-	4 µl template (Consisting of 2 µl H2O and 2 µl of pKJ606 miniprep)<br>	+
-	1/2 µl TAQ Polymerase<br>	+
-	<br>	+
-	PCR Program:<br>	+
	<table style="text-align: left;" border="1" cellpadding="2"		<table style="text-align: left;" border="1" cellpadding="2"
	cellspacing="2">		cellspacing="2">
Line 464:		Line 464:
	<td style="vertical-align: top;">Annealing<br>		<td style="vertical-align: top;">Annealing<br>
	</td>		</td>
-	<td style="vertical-align: top;">55 C<br>	+	<td style="vertical-align: top;">68 C<br>
	</td>		</td>
	<td style="vertical-align: top;">1 min<br>		<td style="vertical-align: top;">1 min<br>
Line 474:		Line 474:
	<td style="vertical-align: top;">72 C<br>		<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top;">2:30 min<br>	+	<td style="vertical-align: top;">4 min<br>
	</td>		</td>
	</tr>		</tr>
Line 482:		Line 482:
	<td style="vertical-align: top;">rep<br>		<td style="vertical-align: top;">rep<br>
	</td>		</td>
-	<td style="vertical-align: top;">29x<br>	+	<td style="vertical-align: top;">44x<br>
	</td>		</td>
	</tr>		</tr>
Line 490:		Line 490:
	<td style="vertical-align: top;">72 C<br>		<td style="vertical-align: top;">72 C<br>
	</td>		</td>
-	<td style="vertical-align: top;">3 min<br>	+	<td style="vertical-align: top;">5 min<br>
	</td>		</td>
	</tr>		</tr>
Line 502:		Line 502:
	</tr>		</tr>
	</table>		</table>
-	<br>	+
-	<br>	+
-	Results: Primers were confirmed working, the next step will be PFU pcr.<br>	+	=== Miniprep of J13002 ===
		+	Date: 3/09<br>
		+	Done by: Tommy & Marie<br>
		+	Methods: Miniprep<br>
		+	Protocols: MP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.1]<br>
		+	Notes: <br>
		+	The minipreps was done according to protocol<br>
		+	Nanodrop:<br>
		+	Tube 1: 38,9 ng/ul, 2nd elution: 20,5 ng/ul <br>
		+	Tube 2: 36,9 ng/ul, 2nd elution: 18,7 ng/ul <br>
		+	Tube 3: 38,4 ng/ul, 2nd elution: 20,0 ng/ul <br>
		+	Tube 4: 32,6 ng/ul, 2nd elution: 16,4 ng/ul <br>
		+
		+	The pooled samples was nanodroped: 20,6 ng/uL

---

Latest revision as of 19:21, 16 October 2010

Lab notes (8/30 - 9/5)

Contents 1 Lab notes (8/30 - 9/5) 1.1 Flagella 1.1.1 Miniprep of "wrong" FlhDCmut 1.2 Photosensor 1.2.1 PCR on pKJ606 with PSfw and VR primers 1.2.2 PCR on pKJ606 with PSfw and PSrv primers 1.2.3 PCR on pKJ606 with fwPS2 and rvPS2 primers 1.3 Retinal 1.3.1 Gradient PCR on ninaB (after PCR with ninaB2fw and ninaB2rv) with ninaBfw and ninaBrv 1.3.2 Gel purification af ninaB from gradient PCR 1.3.3 Gel purification af ninaB from gradient PCR 1.3.4 Restriction digest of ninaB 1.3.5 "Cross-PCR" on ninaB 1.3.6 Miniprep of J13002

Flagella

Since the previous FlhDCmut was wrongly mutated due to incorrect mutation primers, we are now back to square one with new correct primers. The next weeks the flagella-group are working according to the following plan:
1) Miniprep of plasmids with "wrong" FlhDCmut

2) Two-step PCR to get mutatet FlhDC
3) Cut and Ligate into pSB1C3 and pSB1AK3 and transform into TOP10 cells
4) Send to sequencing
5) Characterize biobrick

Miniprep of "wrong" FlhDCmut

Done by: Louise
Date: September 3rd
Protocol: [MP1.2]
Notes:
No changes of protocol were made.
Results: Nanodrop after sample was dried down:
Sample 1: Concentration: 192ng/ul Purity: 1.96/1.90
Sample 2: Concentration: 200ng/ul Purity: 1.84/1.88

The samples were run on a 1.5% gel with a 10kb ladder. The bands are positioned between 1500bp and 1200bp. FlhDCmut in pSB1C3 is 1248bp.

---

Photosensor

PCR on pKJ606 with PSfw and VR primers

Date: 31/8
https://2010.igem.org/wiki/index.php?title=Team:SDU-Denmark/labnotes8&action=edit&section=3 Done by: LC
Methods: PCR
Protocols: CP1.3[1]
Notes:
Premix:
7,5 µl 10xTAQ Buffer
3 µl MgCl2
3 µl VF2
3 µl VR
1,5 µl dNTP
55,5 µl H2O
3 µl template (PS1 miniprep)
3/8 µl TAQ Polymerase

PCR Program:

Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	3 min
Goto2	rep	29x
End	72 C	3 min
Hold	4 C

Results: The bands that showed up were around 2500 BP as expected from the sequencing results. This means that the designed primers have a high possibility of working, so that they will be ordered.

PCR on pKJ606 with PSfw and PSrv primers

Date: 02/09
Done by: LC
Methods: PCR
Protocols: CP1.3[2]
Notes:
Premix:
7,5 µl 10xTAQ Buffer
3 µl MgCl2
3 µl VF2
3 µl VR
1,5 µl dNTP
55,5 µl H2O
3 µl template (PS1 miniprep)
3/8 µl TAQ Polymerase

PCR Program:

Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	2 min
Goto2	rep	29x
End	72 C	3 min
Hold	4 C

Results: The bands that showed up were around 1750 BP, further confirming sequencing results. New primers for taking the whole gene out have been ordered.

PCR on pKJ606 with fwPS2 and rvPS2 primers

Date: 04/09
Done by: LC
Methods: PCR
Protocols: CP1.3[3]
Notes:
Premix:
12,5 µl 10xTAQ Buffer
5 µl MgCl2
5 µl VF2
5 µl VR
2,5 µl dNTP
59,5 µl H2O
4 µl template (Consisting of 2 µl H2O and 2 µl of pKJ606 miniprep)
1/2 µl TAQ Polymerase

PCR Program:

Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	2:30 min
Goto2	rep	29x
End	72 C	3 min
Hold	4 C

Results: Primers were confirmed working, the next step will be PFU pcr.

Retinal

Gradient PCR on ninaB (after PCR with ninaB2fw and ninaB2rv) with ninaBfw and ninaBrv

Date: 30/08
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.1[4]
Notes:
To finde the optimal anneling temperatur a gradient PCR from 60 to 75 degrees
"ninaB" was used as template and ninaB2fw and ninaB2rv was used as primers

PCR Program:

Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	Grad. C	1 min
Elongation	72 C	4 min
Goto2	rep	29x
End	72 C	5 min
Hold	4 C

Vary small amount of the product are observed, it is pooled and gel purifid, the nanodrop shows 372,3 and 377,1 ng/uL.

Gel purification af ninaB from gradient PCR

Date: 31/08
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.1[5]
Notes:
Gel purification was preformed on the pooled product from the gradient PCR according to the protocol
After the gel purification the 2 samples was pooled and nanodroped: 7.0 ng/ul
Furthere PCR was preformed on the purifid product

Gel purification af ninaB from gradient PCR

Date: 1/09
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.3[6]
Notes:
PCR was run according to protocol to the following to this program
Gel purified produckt was used as template and ninaBfw and ninaBrv was used as primers
PCR Program:

Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	Grad. C	1 min
Elongation	72 C	4 min
Goto2	rep	44x
End	72 C	5 min
Hold	4 C

Restriction digest of ninaB

Date: 2/09
Done by: Tommy & Marie
Methods: restriction digest
Protocols: RD1.1[7]
Notes:
EcoRI and PstI ristriction enzymes was used and the ristriction digest was preformed according to protocol
No gel purification was preformed, purificatin was preformed directly from the restriction mixture
Purification was deno using the GFX purification kit and proformed according to that protocol, the purifid product was nanodroped: 3,1 ng/uL

"Cross-PCR" on ninaB

Date: 2/09
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.1[8]
Notes:
Tubes A: ninaBfw and ninaB2rv
Tubes B: ninaB2fw and ninaBrv
NinaB PCR product with ninaB2fw and rv was used as template, both the tubes was run on the same PCR program

Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	68 C	1 min
Elongation	72 C	4 min
Goto2	rep	44x
End	72 C	5 min
Hold	4 C

Miniprep of J13002

Date: 3/09
Done by: Tommy & Marie
Methods: Miniprep
Protocols: MP1.1[9]
Notes:
The minipreps was done according to protocol
Nanodrop:
Tube 1: 38,9 ng/ul, 2nd elution: 20,5 ng/ul
Tube 2: 36,9 ng/ul, 2nd elution: 18,7 ng/ul
Tube 3: 38,4 ng/ul, 2nd elution: 20,0 ng/ul
Tube 4: 32,6 ng/ul, 2nd elution: 16,4 ng/ul

The pooled samples was nanodroped: 20,6 ng/uL