Binding Site Design - October
02/10/2010
- digestion in 50 µl of
- 4x pSB1C3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
- 4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
05/10/2010
- 1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI
- 1= 0S
- 2= 0L
- 3= 10S
- 4= 10L
06/10/2010
20 Miniprep:
Table 1: 20 Minis
| Number | binding site against miRNA x | Position
|
| 1 | 221 | 3.5
|
| 2 | 221 | 1.4
|
| 3 | 1179 | 3.1
|
| 4 | 4286 | 2.6
|
| 5 | 4286 | 4.1
|
| 6 | 4286 | 3.4
|
| 7 | 1179 | 4.4
|
| 8 | 3.5
|
| 9 | 1179 | 1.9
|
| 10 | 221 | 4.6
|
| 11 | 221 | 1.5
|
| 12 | 4286 | 3.5
|
| 13 | 221 | 4.8
|
| 14 | 221 | 2.3
|
| 15 | 4286 | 2.2
|
| 16 | 4286 | 1.3
|
| 17 | 1179 | 2.5
|
| 18 | 1179 | 2.4
|
| 19 | 1179 | 1.5
|
| 20 | 1179 | 4.8
|
- digestion of miMeasure and 6 different synthetic miRNA patterns against endogenous miR122
- gel seperation
- gel purification
- sequencing
12/10/2010
4 miraPCRs for binding sites of miR4286.
- one PCR with an annealing temperature of 45 degree and 16 cycles
- second miraPCR with annealing temperature of 57 degree and also 16 cycles
- nucleotide removal
- digestion
- gel seperation
- gel purification
- ligation in miMeasure and pSB1C3
- transformation
13/10/2010
- pick colonies
- about 8 hours later: do minipreps
- send for sequencing
|
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