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Team:SDU-Denmark/labnotes10
From 2010.igem.org
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
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: 9/14 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)
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