From 2014hs.igem.org
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| | Today we took individual colonies of NEB 10 cells transformed with pLG575 plasmids and put them in LB broth cultures to incubate at 20 degrees C for 24 hours. Growing up these cells will afford us a large supply of the pLG575 plasmids. We ordered more gBlocks from IDT. We will carefully rewrite the protocol for digestion and ligation of the parts that go into the linearized pSB3A1 so transformation can be successful with a future attempt. Also we will run concurrent positive controls in the future to avoid our inability to analyze any future failures. | | Today we took individual colonies of NEB 10 cells transformed with pLG575 plasmids and put them in LB broth cultures to incubate at 20 degrees C for 24 hours. Growing up these cells will afford us a large supply of the pLG575 plasmids. We ordered more gBlocks from IDT. We will carefully rewrite the protocol for digestion and ligation of the parts that go into the linearized pSB3A1 so transformation can be successful with a future attempt. Also we will run concurrent positive controls in the future to avoid our inability to analyze any future failures. |
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| - | <center><b>5/8</b></center> | + | <center><b>5/14</b></center> |
| | + | After very careful preparation, the parts for the expression plasmid were digested, ligated and the resulting ligation product was used to transform NEB 10 cells. Many things were changed from the unsuccessful transformation from last week. The transformed cells are in the 37 degree C incubator so please everyone hold your breath. We worked on the strain 4-1 and 4-2 E coli that will be transformed with two plasmids to produce green and purple fluorescent cells under UV light (for our Human Outreach Illuminarium stand). |
| | + | |
| | + | <center><b>5/15</b></center> |
| | + | The transformation of our expression plasmid was successful! We have the expression plasmid in NEB 10 beta cells on agar supplemented with amp. The next step is to grow up colonies from the agar plate in LB broth. This will give us a large supply of plasmids. We will miniprep the liquid culture getting bare plasmids for contranformation of DB21 DE3 competent cells with both plasmids. Then by using Western blot we can confirm presence of RiAFP in supernatant and lysate. In the ligation step of constructing the expression plasmid, we were very careful to use the correct proportion of DNA from the inserts (Parts A and B) and the vector (linearized pSB1A3). We used 50 ng of vector DNA and made use of a calculator on the NEB web site to calculate the amount of insert DNA to use. There should be a 3 to 1 molar excess of insert DNA to vector DNA to ensure that all the linearized vector is fully occupied by insert DNA. The calculation used the nucleotide lengths of the inserts (about 450 bp for each part) and the vector (2155 bp) to make the calculation. Also for the incubation in the ligation step we incorrectly used 22 degrees C instead of room temperature. This was corrected in the latest transformation. |
| | + | |
| | + | <center><b>5/15</b></center> |
Revision as of 05:37, 12 June 2014
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LAB NOTEBOOK
4/27
This weekend we made stock solutions of antibiotic solutions, prepared sterile LB agar plates supplemented with ampicillin, chloramphenicol, and the combination. We also prepared SOB broth for growing up transformed bacteria.
Consequently, we are ready now to begin assembly of our device.
4/29
Today we transformed competent NEB-10 Beta Cells with pLG575 (secretion plasmid) to have a large supply of plasmid obtainable by mini-prep.
4/30
We took the gBlocks of parts A and B of the expression plasmid and the linearized plasmid pSB3A1 and digested the prefix and suffix of each with restriction enzymes: EchoRI, SpeI, Xbal, PstI, PslI. These enzymes recognize restriction sites and cut them in ways that allow part A to fuse with part B and for the fusion of A-B to fit into pSB3A1. The next step is to permanently ligate the parts so that we end up with a circular plasmid carrying the fusion part. The ligation will be performed on Thursday morning between 10 am and 1 pm.
5/1
Yesterday we cut Parts A and B and pSB3A1 so that they would would together. Today, we ligated the parts into our expression plasmid; T7 promoter RBS RiAFP 6xHis HlyA double terminator. The next step is to transform NEB-10 Beta competent cells with the plasmid.
5/4
We transformed our expression plasmid into NEB-10 Beta cells.
5/6
The transformation failed. We later discovered that we used 0.2 micrograms of plasmid instead of the suggested 1 microgram while constructing the expression plasmid from parts A and B. This altered the volume of the reaction, and was likely the cause of failure.
5/8
Today we took individual colonies of NEB 10 cells transformed with pLG575 plasmids and put them in LB broth cultures to incubate at 20 degrees C for 24 hours. Growing up these cells will afford us a large supply of the pLG575 plasmids. We ordered more gBlocks from IDT. We will carefully rewrite the protocol for digestion and ligation of the parts that go into the linearized pSB3A1 so transformation can be successful with a future attempt. Also we will run concurrent positive controls in the future to avoid our inability to analyze any future failures.
5/14
After very careful preparation, the parts for the expression plasmid were digested, ligated and the resulting ligation product was used to transform NEB 10 cells. Many things were changed from the unsuccessful transformation from last week. The transformed cells are in the 37 degree C incubator so please everyone hold your breath. We worked on the strain 4-1 and 4-2 E coli that will be transformed with two plasmids to produce green and purple fluorescent cells under UV light (for our Human Outreach Illuminarium stand).
5/15
The transformation of our expression plasmid was successful! We have the expression plasmid in NEB 10 beta cells on agar supplemented with amp. The next step is to grow up colonies from the agar plate in LB broth. This will give us a large supply of plasmids. We will miniprep the liquid culture getting bare plasmids for contranformation of DB21 DE3 competent cells with both plasmids. Then by using Western blot we can confirm presence of RiAFP in supernatant and lysate. In the ligation step of constructing the expression plasmid, we were very careful to use the correct proportion of DNA from the inserts (Parts A and B) and the vector (linearized pSB1A3). We used 50 ng of vector DNA and made use of a calculator on the NEB web site to calculate the amount of insert DNA to use. There should be a 3 to 1 molar excess of insert DNA to vector DNA to ensure that all the linearized vector is fully occupied by insert DNA. The calculation used the nucleotide lengths of the inserts (about 450 bp for each part) and the vector (2155 bp) to make the calculation. Also for the incubation in the ligation step we incorrectly used 22 degrees C instead of room temperature. This was corrected in the latest transformation.
5/15
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