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Lab notes (9/13 - 9/19)

Contents 1 Lab notes (9/13 - 9/19) 1.1 Insertion of PS + double terminator in pSB3T5 with J13002 1.1.1 Pfu PCR amplification of PS + double terminator (no.1) 1.1.2 Gel extraction of PS + double terminator 1.1.3 Restriction digest of PS + double terminator, and PSB3T5 no. 1 1.1.4 Pfu PCR amplification of PS + double terminator (no.2) 1.1.5 Gel extraction of PS + double terminator 1.1.6 Restriction digest of PS + double terminator, and PSB3T5 (no.2) 1.1.7 Ligation of PS and pSB1C3 and pCB1AK3 1.1.8 Transfomation of ligated plasmid in Top 10 E.coli

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

Gel extraction of PS + double terminator

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

Gel extraction of PS + double terminator

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.2)

Date: 9/13 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	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.

Results:
DNA conc:

sample	conc. (ng/uL)
PS (used in pSB1C3)	5.5
PS (used in pSB1AK3)	4.2
pSB1C3	6.3
pSB1AK3	14.3

Analysis: the purified DNA was used for ligation.

Ligation of PS and pSB1C3 and pCB1AK3

Date: 9/9 2010
Done By: Maria and Lc
Protocol:LG1.2
Notes:
For each of the ligations three ligation mixtures were prepared. vector concentrations of 10ng/uL (pSB1C3) and 15ng/uL (pSB1AK3) respectively was used for each mixture. Appropiate amount of insert was added to reac vector:insert ratios of 1:1, 1:2 and 1:4 respectively.
Ligation mixtures (PS in pSB1C3):

	L1	L2	L3
T4 ligase buffer	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB1C3	1.5uL	1.5uL	1.5uL
PS	1.5uL	3uL	6uL
H20	14uL	12.5uL	9.5uL

Ligation mixtures (PS in pSB1AK3):

	L1	L2	L3
T4 ligase buffer	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB1C3	1uL	1uL	1uL
PS	2uL	4uL	8uL
H20	14uL	12uL	8uL

The samples was incubated at 17C ON at used for transformation

Transfomation of ligated plasmid in Top 10 E.coli

Date: 9/10 2010
Done By: Maria and Lc
Protocol:CC1.1TR1.1
Notes:
The compotent cells and transformation was carried out according to protocol. Cells transformed with ligations of PS in pSB1AK3 was plated on kanamycine plates.

Results:
the controle plates were okay, and there was many colonies on plates with cells transformed with ligations of pSB1C3 and PS. There was 10-20 colonies on the plates with cells transformed with ligation of PS and pSB1AK3.

Analysis:
The transformation was successfull and colonies was selected and used in coloni PCR.
--Tipi 13:41, 26 September 2010 (UTC)

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