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Team:CIDEB-UANL Mexico/Wet-lab/Notebook
From 2012hs.igem.org
The first day of work in the laboratory, we rehydratated the biobricks 1-6M, 3-12M (corresponding to the stand by construct), 2-12G, 1-10G, 2-14A, 1-10C, 2-11J, 3-12O (corresponding to the low concentration construct) and the 1-14N, 1-2M, 1-6E, 1-10E (corresponding to the high concentration).
The bacterias were transformed with the biobricks mentioned previously and were plated into LB agar with different antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline) depending of the biobrick and placed in the incubator for 24h at 37°C.
Colonies were picked up and culture in test tubes with the antibiotics. In some plates no colonies were observed or almost no one, were re-transformed and plated again for the biobricks 1-14N and 2-11J.
All colonies are ready except for this ones. For Friday is expected that all plates have colonies with all the pieces of the project.
Some colonies were picked up to performance alkaline lysis technique called miniprep miniprep.
More colonies were picked up again because some test tubes get out of the recipient and broke , so were cultured in test tubes, also were made duplicated of the colonies.
Colonies were picked up from an ampicilline plate that were culture the previous day, also were transformed the ones of kanamycin to get the DNA by miniprep.
Due to some DNAs degradated we transformated again the DNAs.
The biobricks were analyzed by electrophoresis using agarose gel 1.2% and then observed using a transiluminator, once the DNA was ready, we made some restriction enzymes cuts of the biobrick pieces and the performance the ligations of each couple of biobricks. For the stand by concentration constructs called L1 is made by the ligation of the biobricks (1-6M and 3-12M), the low concentration construct is made of several constructs called L3 (2-12G and 1-10G), L4 (2-14A and 1-10C) and L2 ( 2-11J and 3-12O) . The last construct is the high concentration made by L6 (1-14N and 1-2M) and the L5 (1-6E and 1-10E)
The ligation of the different constructs prepared in the previous day was transformed also using the same calcium competent cells provided by Biology Development Lab at the Biology Science Faculty.
We obtained colonies only for the constructs L2, L3 and L5, the colonies were picked up into LB medium with the respective antibiotic and incubated at 37°C overnight. New agarose was prepared from SB 20x buffer, the biobricks that were degradated were run again. We tested the voltages in the electrophoresis chamber that had some problems and thought that maybe was the cause of the degradation. Also tested other SB buffers, agarose and chambers and finally found that was our SB buffer we prepared new buffer. We attempted to prepare Ca++ in our lab but couldn’t be finished because of read errors with the spectrum and the freezer conditions.
We prepare the DNA of the tubes with had growing by miniprep. And prepare new material and reagents to attempt again prepare calcium competent in our lab.
We observed in a 1.2% agarose gel the DNA result in the ligations biobricks and then we quantified the DNA using a nanodrop (at the Biology Science Faculty) to know the quantity and performance the restriction enzymes digestion for each clone and determine if have some construct made. We analyzed the digestion of each construct but there were not positive results. New SB 20X buffer was prepared and ligations for each construct were made again.
The new calcium competent bacteria were transformed with the relevant positive control to got the efficiency. We observed the result of the transformation (positive control) 5 colonies and L5 (only got a colony). This results indicate that the bacterias were not enough efficient to get a successful result in the ligation.
We picked up the resulting colonies from L5 made the miniprep and test the SB buffer 1x running a gel with marker to run new and previous cuts. Analyze this DNAs by electrophoresis and then quantify the DNA for the restriction enzymes. We ran the gel and observed that fragments obtained for the L5 ligation did not correspond to the expected. For the next day ligations should be done.
Was transformed the piece 1-14N and took to be plated in plates with kanamycin in the Biology development lab, and prepare more liagtions and transformed again.
The biobrick 1-14N does not grew, we tried three times, changing different conditions but does not work, then a team member investigate about this piece and found that these piece is particularly difficult to get so the best option is use PCR to extract it from a natural DNA sample, order it from a DNA synthesis company, or, for short parts, assemble it from oligos. Only the Ligation 2, 3 and 5 grew, the colonies were picked up in medium and incubated overnight 37°C.
The colonies of the Ligation 2, 3 and 5 did, we used the same ligation and transformed again but this time we use fresh calcium competent prepared in the same day.
Buffer SB 20x was adjusted and finished. New agarose was prepared and discarded the old. Was prepared new buffer 1X from the new 20X. The new transformation with the ligations L2, L3 and L5 grew in the plates and the colonies were picked up and incubated.
We process the colonies incubated and proceed with the DNA extraction by miniprep. The DNA was run in agarose gel 1.2% and then was quantified to performance the characterization by restriction enzymes. The gel of the restriction enzymes was run and the analyzed when were observed that the ligation L3 was positive to the construct delivered a fragment of 1,133 bp, the other construct did not shown a positive result.
We started again Miniprep was made of the parts of the low and high concentration of the project and then ran a 1.2% agarose gel.
Were finished new plates with ampicillin
All BioBrick (except 2-12G and 1-10G the L3 construct) were culture in tubes containing LB medium and plated. Was made a new solution of ethidium bromide with Buffer, 1X buffer was prepared again and also made more miniprep solutions I, II and III.
Today were transformed all the biobricks of the circuit, to have more colonies to continue the project.
