Restriction digest of psiCHECK-2 plasmid
This will be used as backbone for raPCR cloning. Enzymes: XhoI and NotI
Assay:
5 µL 10x NEBuffer 3
5 µL 10x BSA
5 µL plasmid (psiCHECK-2, ~370 ng/µL)
3 µL XhoI
1 µL NotI
18.6 µL H2O
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.
first tries are: hsa-mir-122, hsa-mir122(ran9-12) and mm-mir-376a/375 (Oligos: ra001-003 and ra006)
stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI (ra017/018)
spacer: raPCR_AS13-spacer(0) and raPCR_AS13-spacer(10) (ra012/013)
Oligos were used in standard conc. (100µM)
1. PCR
Oligo
mir-122
mir-122(ran9-12)
mir-375/376a
miR
1 or 3 µL
1 or 3 µL
0.5 or 1.5 µL (each)
spacer(0)or (10)
1 µL
1 µL
1 µL
stop
0 or 0.5 µL each
0 or 0.5 µL each
0 or 0.5 µL each
Total: 12 reactions
each reaction was set up in 30 µL, using 2x Phusion Mastermix for 12 cycles
Temp
Time
95°
05:00
95°
00:30
x 12 cycles
57°
00:45
72°
00:45
4°
forever
PCR purification: each PCR was purified using Qiagen PCR purification Kit and eluted in 32 µL
for the next PCR, three assay will tried:
5µL eluate + 1 µL of each stop oligo in 50µL total volume
5µL eluate + 2 µL of each stop oligo in 50µL total volume
20µL eluate + 1 µL of each stop oligo in 50µL total volume
stop-oligos: raPCR_AS13-stop_rev_NotI and raPCR_AS13-stop_fw_XhoI
2. PCR
In total there were 72 reactions. Each was run with 2x Phusion Mastermix, missing volume was filled with water.
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