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:

PCR program:

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:

Restriction mixture PS + double terminator:

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:

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:

PCR program:

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: 9/14 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:

Restriction mixture PS + double terminator:

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:

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:

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):

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)

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