Lab notes (9/13 - 9/19)
Insertion of PS + double terminator in pSB3T5 with J13002
Date: 9/13 - 9/16 2010
Done By: Maria and Lc
Protocol: CP1.1DE1.3RD1.1LG1.2CC1.1TR1.1CP1.3
Pfu PCR amplification of PS + double terminator (no.1)
Date: 9/13 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
6 PCR reactions are prepared. 3.5uL Miniprep of PS+ double terminator in pSB1AK3 are diluted in 3.5uL H2O to reach a 2x dilution, and are used as template. PCR tubes are marked PS+B0015.1 A-F.
Premix x6:
pfu buffer + MgSO4 |
35uL |
dNTP mix |
10.5uL |
VF2 primer |
10.5uL |
VR primer |
10.5uL |
H20 |
273uL |
pfu polymerase |
3uL |
PS + Double terminator in pSB1AK3 (2x diluted) |
7uL |
PCR program:
start |
95C |
3min |
denaturating |
95C |
2min |
annealing |
55C |
30s |
elongation |
72C |
4min30s |
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. 2500bp and bands were extracted by gel extraction
Date: 9/13 2010
Done By: Maria and Lc
Protocol:DE1.3
Notes:
DNA was extracted from gel according to protocol and each sample was diluted in 20uL.
Analysis:
samples were pooled and used for restriction digest.
Restriction digest of PS + double terminator, and PSB3T5 no. 1
Date: 9/13 2010
Done By: Maria and Lc
Protocol:RD1.1DE1.3
Notes:
Restriction mixture pSB3T5:
H2O |
12uL |
FD green buffer |
2uL |
SpeI |
1uL |
PstI |
1uL |
pSB3T5 |
5uL |
Restriction mixture PS + double terminator:
H2O |
38uL |
FD green buffer |
8uL |
XbaI |
4uL |
PstI |
4uL |
PS + double terminator |
30uL |
Digested samples were loaded onto a 1.5% agarose extraction gel. Uncut PS + double terminator and pSB3T5 were used as controles. Gene ruler DNA ladder mix was used as marker.
Loading scheme:
Lane |
sample |
1 |
PS + double terminator |
2 |
uncut PS + double terminator |
3 |
pSB3T5 |
4 |
uncut pSB3T5 |
5 |
marker |
DNA was extracted from gel, however undiluted washing buffer was used and the DNA in the samples was destroyed.
Pfu PCR amplification of PS + double terminator (no.2)
Date: 9/13 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
6 PCR reactions are prepared. 3.5uL Miniprep of PS+ double terminator in pSB1AK3 are diluted in 3.5uL H2O to reach a 2x dilution, and are used as template. PCR tubes are marked PS+B0015.1 A-F.
Premix x6:
pfu buffer + MgSO4 |
35uL |
dNTP mix |
10.5uL |
VF2 primer |
10.5uL |
VR primer |
10.5uL |
H20 |
273uL |
pfu polymerase |
3uL |
PS + Double terminator in pSB1AK3 (2x diluted) |
7uL |
PCR program:
start |
95C |
3min |
denaturating |
95C |
2min |
annealing |
55C |
30s |
elongation |
72C |
4min30s |
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. 2500bp and bands were extracted by gel extraction
Date: 9/14 2010
Done By: Maria and Lc
Protocol:DE1.3
Notes:
DNA was extracted from gel according to protocol and each sample was diluted in 20uL.
Analysis:
samples were pooled and used for restriction digest.
Restriction digest of PS + double terminator, and PSB3T5 (no.2)
Date: 9/14 2010
Done By: Maria and Lc
Protocol:RD1.1DE1.3
Notes:
Restriction mixture pSB3T5:
H2O |
14uL |
FD green buffer |
4uL |
SpeI |
2uL |
PstI |
2uL |
pSB3T5 |
20uL |
Restriction mixture PS + double terminator:
H2O |
38uL |
FD green buffer |
8uL |
XbaI |
4uL |
PstI |
4uL |
PS + double terminator |
30uL |
Digested samples were loaded onto a 1.5% agarose extraction gel. Uncut PS + double terminator and pSB3T5 were used as controles. Gene ruler DNA ladder mix was used as marker.
Loading scheme:
Lane |
sample |
1 |
Marker |
2 |
uncut PS + double terminator |
3 |
PS + double terminator |
4 |
uncut pSB3T5 |
5 |
pSB3T5 |
DNA was extracted from gel, according to protocol. Samples were diluted in 20uL H2O. 2. dilution of PS + doubletermiantor was diluted in 10uL H2O
Results:
DNA conc:
sample |
conc. (ng/uL) |
PS + double terminator (1. dilution) |
4.8 |
PS + double terminator (2. dilution) |
2.3 |
pSB3T5 |
26.4 |
Analysis:
the purified DNA was used for ligation.
Ligation of PS + double terminator and pSB3T5
Date: 9/14 2010
Done By: Maria and Lc
Protocol:LG1.2
Notes:
Three ligation mixtures was prepared. vector concentrations of 25ng/uL was used for each mixture. Appropiate amount of insert was added to reac vector:insert ratios of 1:1, 1:2 and 1:3 respectively.
Ligation mixtures (PS + double terminator in pSB3T5):
|
L1 |
L2 |
L3 |
T4 ligase buffer |
2uL |
2uL |
2uL |
T4 ligase |
1uL |
1uL |
1uL |
pSB3T5 |
1uL |
1uL |
1uL |
PS + double terminator |
8uL (2. dilution) |
7uL |
11uL |
H20 |
8uL |
9uL |
5uL |
The samples were incubated at 17C ON at used for transformation
Transfomation of ligated plasmid in Top 10 E.coli
Date: 9/15 2010
Done By: Maria and Lc
Protocol:CC1.1TR1.1
Notes:
The compotent cells and transformation was carried out according to protocol.
Results:
the controle plates were okay, and colonies of different sizes was observed on plates of cells transformed with ligations.
Analysis:
The transformation was successfull and colonies was selected and used in coloni PCR.
--Tipi 13:41, 26 September 2010 (UTC)
Transfomation of pKJ606 in Mg 1655 E.coli
Date: 9/15 2010
Done By: Maria and Lc
Protocol:CC1.1TR1.1
Notes:
The compotent cells and transformation was carried out according to protocol.
Results:
the controle plates were okay, and large colonies was observed on the plates.
Analysis:
The transformation was successfull and colonies was selected and used in coloni PCR.
--Tipi 05:48, 28 September 2010 (UTC)