Team:SDU-Denmark/labnotes8

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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

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.
Team SDU-Denmark Miniprep af flhDCmut i 1C3.jpg


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.
Team-SDU-Denmark-PSfwVR.jpg

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.
Team-SDU-Denmark-PSfwrv.jpg

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.

Date: 30/08
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.3[4]
Notes:
To finde the optimal anneling temperatur a gradient PCR from 60 to 75 degrees

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

Colonies 34, 37, 61 and 63 was selected for minipreps and freezing cultures.