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Lab notes (9/6 - 9/12)

Contents 1 Lab notes (9/6 - 9/12) 1.1 Flagella Group 1.1.1 Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3 1.1.2 Ligation of FlhDCmut and pSB1C3 1.1.3 Ligation of FlhDCmut and pSB1AK3 1.2 Photosensor group 1.2.1 Taq gradient PCR of pKJ606 with PS primers 1.2.2 Insertion of PS in pSB1C3 and pSB1AK3 1.2.2.1 Pfu PCR amplification of PS (no.1) 1.2.2.2 Pfu PCR amplification of PS (no.2) 1.2.2.3 Gel extraction of PS

Flagella Group

Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3

Date: 9/12 2010
Done By: Sheila
Protocol: RD1.1
Notes: The restriction digests are made in preperation for two different ligations, the first is the ligation of FlhDCmut into the submission backbone; pSB1C3. The second is the the ligation of FlhDCmut into pSB1AK3. Different restriction enzymes were used in, as shown in the following tables

Restriction Digest mixtures
FlhDCmut and pSB1C3 Restriction digest
FlhDCmut

H2O	48μL
EcoR1	4μL
Pst1	4μL
FD Green buffer	8μL
Sample	20μL

pSB1C3

H2O	24μL
EcoR1	2μL
Pst1	2μL
FD Green buffer	4μL
Sample	10μL

FlhDCmut and pSB1AK3 Restriction digest
FlhDCmut

H2O	48μL
EcoR1	4μL
Spe1	4μL
FD Green buffer	8μL
Sample	20μL

pSB1AK3

H2O	24μL
EcoR1	2μL
Xba1	2μL
FD Green buffer	4μL
Sample	10μL

Following digestion, the samples were loaded and run on a 1.5% agarose gel, before being extracted prior to ligation.

Results:

Ligation of FlhDCmut and pSB1C3

Date: 9/12 2010
Done By: Sheila
Protocol: LG1.2
Notes:
Results:

Ligation of FlhDCmut and pSB1AK3

Date: 9/12 2010
Done By: Sheila
Protocol: LG1.2
Notes:
Results:

Photosensor group

Taq gradient PCR of pKJ606 with PS primers

Date: 9/7 2010
Done By: Maria and Lc
Protocol: CP1.3
Notes:
A gradient PCR was carried out to determine the appropiate annealing temperature of the new PS primers. PCR tubes were marked PSII A-H.

Premix x 9

Taq buffer (10x)	22.5uL
MgCl2	9uL
PSII fw primer	9uL
PSII rv primer	9uL
dNTP mix	4.5uL
H20	158.5uL
Taq polymerase	1uL

miniprep of pKJ606 was used as template. 1uL was distrubuted into each tube.
PCR program:

start	95C	2min
denaturating	95C	1min
annealing	se additional table	1min
elongation	72C	2min
go to	2	rep.29x
end	72C	5min
hold	4C

Anealing temperatures:

56.5C

58.3C

60.7C

63.3C

66C

68.6C

71C

73C

PCR product was loaded onto a 1.5 agarose gel. gene ruler DNA ladder mix (red) was used as marker.

Results:

Analysis:
Only very weak bands was observed at app. 2000 bp, and very strong bands were observed at app. 4000 bp, indicating that too much template was used in the PCR. An additional PCR was carried out using diluted miniprep as template.
--Tipi 10:27, 21 September 2010 (UTC)

Insertion of PS in pSB1C3 and pSB1AK3

Date: 9/8 - 9/12 2010
Done By: Maria and Lc
Protocol: CP1.1DE1.3RD1.1LG1.2CC1.1TR1.1CP1.3

Pfu PCR amplification of PS (no.1)

Date: 9/8 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
5 PCR reactions are prepared. 2uL Miniprep of pKJ606 are diluted in 8uL H2O to reach a 5x dilution, and are used as template. PCR tubes are marked PSIII A-E.
Premix x6:

pfu buffer + MgSO4	30uL
dNTP mix	9uL
PSII fw primer	9uL
PSII rv primer	9uL
H20	230uL
pfu polymerase	2.5uL
pKJ606 miniprep (5x diluted)	10uL

PCR program:

start	94C	3min
denaturating	94C	2min
annealing	68C	30s
elongation	72C	2min30s
go to	2	rep.29x
end	72C	5min
hold	4C

All of the PCR product was loaded onto a 1.5 agarose extraction gel. Gene ruler DNA ladder mix was used ad marker.

Results:
Analysis:
No bands were visible in the gel, indicating that the PCR reaction had been unsuccessfull. This could be due to the low elongation time, wherefore another PCR reaction with a longer elongation time was carried out.

Pfu PCR amplification of PS (no.2)

Date: 9/9 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
5 PCR reactions are prepared. 2uL Miniprep of pKJ606 are diluted in 8uL H2O to reach a 5x dilution, and are used as template. PCR tubes are marked PSIV A-E.
Premix x6:

pfu buffer + MgSO4	30uL
dNTP mix	9uL
PSII fw primer	9uL
PSII rv primer	9uL
H20	228uL
pfu polymerase	2.5uL
pKJ606 miniprep (5x diluted)	12uL

PCR program:

start	94C	3min
denaturating	94C	2min
annealing	68C	30s
elongation	72C	4min20s
go to	2	rep.29x
end	72C	5min
hold	4C

All of the PCR product was loaded onto a 1.5 agarose extraction gel. Gene ruler DNA ladder mix was used ad marker.

Results:
Analysis:
Bands were observed at app. 2000bp and bands were extracted by gel extraction

Gel extraction of PS

Date: 9/9 2010
Done By: Maria and Lc
Protocol:CP1.1

Notes: