Team:SDU-Denmark/labnotes8

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__TOC__
__TOC__
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== Photosensor ==
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=== PCR on pKJ606 with PSfw and VR primers ===
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Date: 31/8<br>
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Done by: LC<br>
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Methods: PCR<br>
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Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
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Notes: <br>
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Premix:<br>
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7,5 µl 10xTAQ Buffer<br>
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3 µl MgCl2 <br>
 +
3 µl VF2 <br>
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3 µl VR <br>
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1,5 µl dNTP<br>
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55,5 µl H2O<br>
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3 µl template (PS1 miniprep)<br>
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3/8 µl TAQ Polymerase<br>
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<br>
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PCR Program:<br>
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<table style="text-align: left;" border="1" cellpadding="2"
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cellspacing="2">
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<tr>
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<td style="vertical-align: top;">Start<br>
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</td>
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<td style="vertical-align: top;">94&nbsp; C<br>
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</td>
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<td style="vertical-align: top;">2 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Denaturing<br>
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</td>
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<td style="vertical-align: top;">94 C<br>
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</td>
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<td style="vertical-align: top;">1 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Annealing<br>
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</td>
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<td style="vertical-align: top;">55 C<br>
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</td>
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<td style="vertical-align: top;">1 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Elongation<br>
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</td>
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<td style="vertical-align: top;">72 C<br>
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</td>
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<td style="vertical-align: top;">3 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Goto2<br>
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</td>
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<td style="vertical-align: top;">rep<br>
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</td>
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<td style="vertical-align: top;">29x<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">End<br>
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</td>
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<td style="vertical-align: top;">72 C<br>
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</td>
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<td style="vertical-align: top;">3 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Hold<br>
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</td>
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<td style="vertical-align: top;">4 C<br>
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</td>
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<td style="vertical-align: top;"><br>
 +
</td>
 +
</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.
=== Flagella ===
=== Flagella ===
<br>
<br>

Revision as of 13:21, 26 September 2010

Lab notes (8/30 - 9/5)

Contents

Photosensor

PCR on pKJ606 with PSfw and VR primers

Date: 31/8
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.

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

Two-step PCR of miniprep


First step: PCR of miniprep with mutation primers


Done by: Louise
Date: September 7th
Protocol: [CP1.1]
Notes:
3 x Premix 1:
114.3ul water
15ul Pfu Buffer
4.5ul dNTP
3ul MgSO4
4.5ul FlhDC fw
4.5ul FlhDCmut rev
1.2ul PFU
3ul template (miniprep)

3 x Premix 2:
114.3ul water
15ul Pfu Buffer
4.5ul dNTP
3ul MgSO4
4.5ul FlhDCmut fw
4.5ul FlhDC rev
1.2ul PFU
3ul template (miniprep)

Results:
The PCR samples were run on a 1.5% gel with a 100bp-1000bp ladder. All samples looked okay and were pooled as sample 1.1 and sample 2.1
NanoDrop:
Sample 1.1: Concentration: 359.5ng/ul Purity: 1.83/2.20
Sample 2.1: Concentration: 304.1ng/ul Purity: 1.85/2.23

Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers


Done by: Maria
Date: September 9th
Protocol: [CP1.1]
Notes:
Premix:
38ul water
5ul PFU buffer + MgSO4
1.5ul dNTP
1ul Sample 1.1
1ul Sample 2.1
1.5ul FlhDC fw primer
1.5ul FlhDC rev primer
0.5ul PFU

PCR Program:

1:Start
 95C
 2 min
2: Denaturing
 95C
 30 sec
3: Annealing
 56C
 30 sec
4: Elongation
 72C
 2 min
5:
 GO TO
 2 rep. 4x
6: Denaturing
 95C
 30 sec
7: Annealing
 63C
 30 sec
8: Elongation
 72C
 2 min
9:
 GO TO
 6 rep. 25x
10: End
72C
 5 min
12: Hold
 4C


Results:
Some of the PCR product was run on a 1.5% gel with a 10kb ladder.
The rest of the product was extracted from a new gel.

PCR of Gel extraction


Done by: Maria
Date: September 9th
Protocol: [CP1.1]
Notes:
Premix x 6:
234ul water
30ul PFU buffer + MgSO4
9ul dNTP
9ul FlhDC fw primer
9ul FlhDC rev primer
2.5ul PFU
6ul template

1:Start
 95C
 2 min
2: Denaturing
 95C
 30sec
3: Annealing
 63C
 30 sec
4: Elongation
 72C
 2 min
5:
 GO TO
 2 rep. 29x
6: End
72C
 5 min
7: Hold
 4C


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