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From 2010.igem.org

Lab notes (8/30 - 9/5)

Contents 1 Lab notes (8/30 - 9/5) 1.1 Flagella 1.1.1 Miniprep of "wrong" FlhDCmut 1.1.2 Two-step PCR of miniprep 1.1.2.1 First step: PCR of miniprep with mutation primers 1.1.2.2 Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers 1.1.2.3 PCR of Gel extraction

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

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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
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

1:Start	94C	2 min
2: Denaturing	94C	1 min
3: Annealing	55C	1 min
4: Elongation	72C	1 min 30 s
5:	GO TO	rep. 29x
6: End	72C	3 min
7: Hold	4C