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Team:Tuebingen/NotebookReports
From 2012.igem.org
Contents |
Weekly Reports
Week 1 (7/9-7/15)
Thursday the 12th of July was the first day in our laboratory.
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.
2. To determine the optimal annealing temperature, a gradient PCR for the parts 1-8 was performed. Doing a gel-electrophoresis the PCR results were tested.
3. Preparing chemocompetent E. coli TOP 10 cells after Inoue protocol.
Week 2 (7/16-7/22)
1. A successful PCR for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a gel-electrophoresis.
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.
2. To test the competence of the chemocompetent E. coli TOP 10 cells a transformation with pRS313 and a negative control was done. Due to the fact that the competent cells didn’t work, new chemocompetent E. coli TOP 10 cells were prepared. The Transformation of pRS313, pRS315 and pRS316 in these competent cells was successful.
Week 3 (7/23-7/29)
1. The first ligation of the parts 1-8 in pGEM with following transformation in the competent E. coli TOP10 was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. The restriction digest was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gelelectrophoresis showed that the ligation was not successful, because only one band of 3000bp for the pGEM vector was visible.
2. A new PCR of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product 8 was purified. The PCR-product 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.
Week 4 (7/30-8/05)
1. The shipment with the synthesized parts (mPRq of Danio rerio and MIG1) arrived.
2. A transformation of the parts mPRq and MIG1 was performed using the competent E. coli TOP10 cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful. The first attempt to isolate the plasmids was with usage of a plasmid preparation kit. But this try failed. Therefore the plasmid isolation was successfully repeated using the alkaline lysis.
3. A new PCR of the parts 1-8 with Taq/Pfu-polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.
Week 5 (8/06-8/12)
1. A small restriction digest of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the right overhangs for a ligation to take place later. The restriction digest was executed with the parts Mig1 and mPR of Danio rerio, too. Due to unclean plasmids and DNA (perhaps to much salts in the tubes) this step had to be repeated several times, because the restriction digest being incomplete.
Therefore the plasmids (pRS313, 315, 316 and the parts Mig1, mPR D.R.) were purified again with a midi DNA purification kit. Now the restriction digest was executed completely.
2. A new PCR of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR-product. The polymerase Herculase was used due to its preciseness and productivity. Indeed the result of the PCR was better than with Pfu/Taq-polymerase.
Week 6 (8/13-8/19)
1. The Phenol/Chloroform filtration of pRS 313, pRS 315, pRS 316, mig1, mPR d.R. was completed. Concentration was measured afterwards. One new petri dish with colonies of parts 1 to 8 was established.
2. Preparative gel for PCR products 3,5 and 5 (from PCR with Herculase) delivered new template for another PCR.
3. mini-Plasmid preparation of parts 1 to 8 was executed, the purified DNA was digested afterwards. An analytical gel was used to check for inserts. Result: It was not arbitrable, whether inserts existed or not. A new SP6/T7 PCR was performed on Wednesday.
4. PCR with primers SP6/T7 was performed since the promoters fit to the multiple cloning site of pGEM T-vector. The liquid cultures of pRS313, pRS315, pRS316, mig1, mPR D.r. were prepared, incubation over night.
5. Midi-Prep of pRS313, pRS315, pRS316, mig1, mPR D.r. was performed and new chemo-competent cells were raised. New PCR with template 3,4,5.1 from Herculase PCR from 10.8 was executed. Taq and Taq/Pfu were used as Polymerase.
6. Sequences were interpreted and a test transformation with new chemo-competent cells was executed. Ligation of 3, 6, 7, 8 in pGEM vector was performed. Reaction took place over night at 4 °C (39.2 Fahrenheit). Additionally, a PCR purification of parts 3,5 was executed. Finally we preformed a Midi-Prep for mPR d.r and mPR X.I.
Week 7 (8/20-8/26)
1. New culture of part 4 was established to perform plasmid preparation, the ligation of 3,6,7,8 from last week was transformed in E.Coli, TOP10 strain.
2. PCR of parts 1 and 2 with herculase, it was checked with analytical gel afterwards.
3. Since we ran out of lacZ,luciferase DNA, we decided to transform remaining DNA of lacZ, luciferase into E.coli. Transformation was successful. The purified PCR products of parts 5 from last week were joined due to low concentration. A precipation with ethanol was performed afterwards to increase concentration even further. The concentration of the DNA was exactly zero, so it did not seem to work.
4. Colony-PCR of cultures from parts 3,6,7,8 (5 colonies each) was performed at 50 °C (122 degrees Fahrenheit) to check whether the correct insert was integrated into the plasmids. An analytical gel revealed that the colony-PCR didn’t work.
5. Transformation of ligation of part 5 into E.coli. The restriction digestion of part Part 9,10,11 and pRS 313, 315, 316 was checked with an analytical gel.
6. Mini plasmid preparation of parts 3,6,7,8 with subsequent restriction digestion. Positive samples were prepared for sequencing. New PCR of part 5 with purified PCR product as template.
7. Great restriction digestion of plasmids pRS 313, 315, 316 and mPR d.r, mPR x.l , mig1 with XbaI and SpeI. The parts mig1, mPR D.r. und mPR X.l. were unstiched with an preparative gel ad purified afterwards.
8. Mini plasmid preparation of parts 6 and 7 (5 colonies each) and tests for inserts. Ligation of parts 9,10,11 in iGEM vector.
