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Team:Heidelberg/Notebook/miMeasure/September
From 2010.igem.org
13/09/2010
Assay:
Restriction digest was performed for approx. 5h raPCR to create binding sites for different miRNAs This random assembly PCR (raPCR) will be done to create binding site patterns for the miRNAs mentioned. In the first PCR step the oligos will basically anneal and constructs of different lengths will form. In the second step, the stop oligos are used as primers to amplify the previously formed constructs.
stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI (ra017/018) Oligos were used in standard conc. (100µM)
for the next PCR, three assay will tried:
stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI
In total there were 72 reactions. Each was run with 2x Phusion Mastermix, missing volume was filled with water.
DNA was stored in fridge afterwards 14/09/201072 PCRs were analyesed on 1% agarose gel Best looking lanes of spacer(0) and spacer(10) were choosen for preparative gel preparative gel (1.5%) was made for miR122 Sample for small Binding site patterns (100-250 bp) and large patterns (250-400 bp) were taken. each were eluted in 1 column (a lot of work for gel extraction...)
15/09/2010Gel extracted samples were digested with Not/Xho for cloning into psiCheck-2 vector: 5µL template in total 30µL using 1µL xho and 0.6µL Not enzyme digest was then purified using qiagen nucleotide removal kit, elution in 30µL due to a problem of the vector-size (it has 8000 instead of 6000 bp), ligation was set-up overnight at roomtemperature ligation: 2µL buffer 1µL ligase 7µL water 1µL backbone (6000bp) 9µL restriction digest
16/09/2010Transformation of ligations: 5µL ligation assay in 50µL TOP10 E.coli 25 min on ice 45sec heat shock on 42°C 1.5-2h shaking at 37°C plated 200µL on Amp-Plates after incubating ~8h, at 37°C, the plates were incubated overnight at room temperature 17/09/2010Colonies were visible in reasonable numbers on every plate colony pcr was performed to check clones 20µL assay PCR assay, using 2x PCR master mix (Taq) from Fermentas one colony was dissolved in 20µL water 5µL of this bacteria solution was used as PCR template PCR conditions as recommended from Fermentas Minipreps will be setup in the evening for overnight cultures (4mL, Amp) 5 for each construct 28/09/2010
29/09/2010
30/09/2010
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