Team:SDU-Denmark/labnotes8

From 2010.igem.org

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=== Flagella ===
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<br>
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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: <br>
 +
1) Miniprep of plasmids with "wrong" FlhDCmut <br><br>
 +
2) Two-step PCR to get mutatet FlhDC<br>
 +
3) Cut and Ligate into pSB1C3 and pSB1AK3 and transform into TOP10 cells <br>
 +
4) Send to sequencing <br>
 +
5) Characterize biobrick <br><br>
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==== Miniprep of "wrong" FlhDCmut ====
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<br>
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''Done by:'' Louise <br>
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''Date:'' September 3rd <br>
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''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2]] <br>
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''Notes:'' <br>
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No changes of protocol were made. <br>
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''Results:''
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Nanodrop after sample was dried down: <br>
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'''Sample 1:''' Concentration: 192ng/ul Purity: 1.96/1.90 <br>
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'''Sample 2:''' Concentration: 200ng/ul Purity: 1.84/1.88 <br><br>
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----
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==== Two-step PCR of miniprep ====
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<br>
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===== First step: PCR of miniprep with mutation primers =====
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<br>
 +
''Done by:'' Louise <br>
 +
''Date:'' September 7th <br>
 +
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
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''Notes:'' <br>
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'''3 x Premix 1:''' <br>
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114.3ul water <br>
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15ul Pfu Buffer <br>
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4.5ul dNTP <br>
 +
3ul MgSO4 <br>
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4.5ul FlhDC fw <br>
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4.5ul FlhDCmut rev <br>
 +
1.2ul PFU <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>
 +
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 <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>
 +
 +
===== Second 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>
 +
Start:

Revision as of 13:23, 23 September 2010

Lab notes (8/30 - 9/5)

Contents


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: 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:
Start:

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