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Team:DTU-Denmark-2/Notebook/Lab notes
From 2011.igem.org
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Lab notes |
JulyWeek 1: 04.07.2011 - 10.07.2011First week! Time was spent on designing the Plug 'n' Play assembly system, figuring out what 48 biobricks to construct, and searching for appropriate DNA templates.Week 2: 11.07.2011 - 17.07.2011WednesdayThe first batch of primers have been ordered, so we are waiting in excitement for them to arrive, so we can start our work in the lab.Week 3: 18.07.2011 - 24.07.2011TuesdayFirst day in the lab and 48 biobricks to go. This is exciting!We plan to construct 48 new BioBricks. We have now established 17 of them and ordered primers for them with USER tails. We started the day with 11 PCR reactions. 6 of them were correct, and they were purified with a purification Kit (GE Healthcare). Only 42 biobricks to go! Biobricks in the bag:
ThursdayThe failed PCR reactions from tuesday were repeated. This time we used a different annealing temperature, and....IT WORKED!New biobricks in the bag:
The fragments will be frozen and later purified. We still have not succeed to make BioBricks pyrG and pyrG-DR (used as a marker in fungi), despite different PCR attempts with a high annealing (59°) and low (56°) temperature. Work for iGEM Copenhagen team We have established collaboration with the iGEM team from Copenhagen University. We will costumize a number of their BioBricks to use with our Plug 'n' Play system. This is a great way of showing how easy it is to costumize the Plug 'n' Play system for any application, and the Copenhagen team will save a lot of time. Today we transformed competent E.coli cells with some of the standard biological parts from iGEM, BBa_R0010, Bba_B0015 and BBa_J52034, and later we will make them fit the Plug 'n' Play with DNA system. FridayWe made a PCR of a troublesome BioBrick again - but this time with a small modification. We made a PCR mix with and without MgCl2 50mM in the reactions. And...IT WORKED with MgCl2 50mM!Biobrick obtained:
The fragments will be frozen and later purified . Week 4: 25.07.2011 - 31.07.2011MondayWe got a new delivery of primers from IDT today. They are to be diluted and stocked, before use. We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.The PCR reactions were successful, and following biobricks are ready for Plug'n'Play:
TuesdayThe primers recieved yesterday will be used for amplification of many many many new biobricks today.Out of 15 PCR reactions 5 succeeded:
WednesdayToday we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.One succeeded:
AugustWeek 5: 01.08.2011 - 07.08.2011MondayNew week, new energy, and lots of team spirit! Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!New biobricks in the bag:
WednesdayThere are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR.Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened. Thursday22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved! We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section. Week 5: 08.08.2011 - 14.08.2011MondayToday we made the first USER cloning, we are very excited to see if the system works the way, we want it to!All our biobricks are divided into devices, see protocol for USER MIX. The following devices were cloned today:
PCR reactions done today:
After recieving the new DNA template we finally succeeded in getting biobricks pyrG and pyrG-DR. The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration. TuesdayE.coli was inoculated with the plasmides done by USER cloning yesterday.PCR done today:
Purification with GFX kit of the following biobricks:
USER cloning done today:
WednesdayThe U2OS cells was investigated in the microscope. They had a confluency of about 90%, an therefore ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")FridayToday 13 PCR reaction were carried out, some parts were for characterisation of two promoters: pAlc and DMKP-P6, and the rest were parts for the Copenhagen iGEM team.
The U2OS cells were splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday. Week 7: 15.08.2011 - 21.08.2011MondayThe U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They were placed in the incubator overnight - and will be ready for transfection with plasmides tomorrow.In preparation for characterization of two promoters with β-galactosidase assay the plasmid p68 cut with restriction enzyme. Purification of PCR products with GFX kit. 9 PCR reactions were done today
USER cloning and transformation in E.coli today:
TuesdayWe transfected U2OS cells with plasmides containing GFP and placed them in the incubator overnight.USER cloning and transformation in E.coli today:
WednesdayInoculations of E.coli transformants with USER devices cloned yesterday. Restriction analysis of device 4, 7, 8, and 27. USER cloning and transformation in E.coli today:
Mini-prep purifications and restriction analysis of plasmids from transformed E.coli . ThursdayThe coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.Transformation of Aspergillus nidulans for promotor characterization. FridayUSER cloning and transformation in E.coli today:
Week 8: 22.08.2011 - 28.08.2011MondayThe U2OS cells were splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.TuesdayThe U2OS cells were transfected with all the mammalian plasmides.Hopefully, we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.Measurements of plasmids containing our devices before sending them to DNA Technology for sequencing. The measurements were done with Qubit Fluorometer from Invitrogen. More restriction analysis of plasmids were done. WednesdayThe coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great -everything worked = we are done with the mammalian cells. Hip hip hurra!Transformation of Aspergillus nidulans for proof of concept. SeptemberWeek 9: 29.08.2011 - 04.09.2011TuesdayPlasmids and primers were sent to DNA Technology for sequencing.WednesdayPCR reaction of the shipping plasmid were done. USER tails were applied to the shipping plasmid, so it can be easy and quick assembled with the 2 BioBricks and the device.
ThursdayUSER cloning of the two promoters and device 12 with the shipping plasmid were done.Due to the extensive use of biobrick plasmid_fun(28) and plasmid_mam(30) we made some more. Week 10: 05.09.2011 - 11.09.2011MondayRestriction analysis of shipping plasmid was made to ensure that the USER cloning were done correctly. The shipping plasmid was sent to sequencing.Inoculation of fungi for β-galactosidase, Bradford assay and Fluorescence microscope. TuesdayToday we checked out our fungi in fluorescence microscope, a beautiful sight it was! We have now proven that our system works in fungi as well as in mammalian cells.WednesdayA whole day was spent in the lab doing the β-galactosidase and Bradford assay for characterisation of our two promoters.FridayThis might be one of the last iGEM days in lab. We are measuring the DNA concentration of the biobricks, so they can be sent to MIT next week.OctoberNovember |
