All Times in Zulu
Zulu Time All.
14-6-17
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| id | 6-17 |
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| Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Begun 6-17) |
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Stock Concentrations: EBFP2: 1118 ng/µL MAV: unknown. 1856: Diluted DNA samples for both x100 before transformation 4 Plates, LO HI for both. 2144: Culture plates incubated overnight at 30°C for 16-18 hours. |
14-6-18
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| id | 6-18 |
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| Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Begun 6-17) |
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| 1426: Checked cell plates. EBFP2 produced colonies. MAV1212 overgrown. |
| MAV1212 Streaking (Start 6-18) | Propagating EBFP2: Pick Colonies (Start 6-17) |
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| 2044: Picked single colonies from each plate and streaked over xgal plates. Let on the incubator overnight at 30°C for 16-18 hours. | 2040: Picked one colony each from the HI and LO plates and immersed in LB Media. Left on the shaker incubator overnight. |
14-6-19
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| id | 6-19 |
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| MAV1212 Streaking (Start 6-18) OVER |
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| 1254: Disaster. No colonies. |
| Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Start 6-19) |
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Concentrations: EBFP2: 12 ng/µL MAV: 4 ng/µL
4 Plates, LO HI for both. 1715: Culture plates inseerted into 30°C incubator. |
14-6-20
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| id | 6-20 |
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| Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Start 6-19) OVER |
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1105: Low transformation rate. Suspicions fall on faulty plates. Plating to be redone later with existing samples and streaking. |
14-6-23
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| id | 6-23 |
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| Propagating MAV1212: Colony Picking (Start 6-17) | Propagation MAV1212 (Start 6-23) |
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Attempted to isolate monoclonal colony from MAV1212 lawn. Inserted into Amp+ LB medium. Left on incubator shaker at 2114. | Re-plated transformation suspension on Amp+ plates. Streaked colonies from lawn. Left on 30°C incubator at 2120. |
14-6-24
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| id | 6-24 |
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| Propagating MAV1212: Colony Picking (Start 6-17) OVER | Propagation MAV1212 (Start 6-23) OVER |
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Medium Clear. Growth failed. | Current MAV1212 vectors abandoned due to overactive ccdB gene killing off cells. Awaiting arrival of new vectors with better survivability. |
| Propagation MAV1212-EBFP2 (Start 6-24) |
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Transformed onto Amp+ plates. Left on 30°C incubator at 2135. |
14-6-25
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| id | 6-25 |
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| Propagation MAV1212-EBFP2 (Start 6-24) | Propagation hEF1a (Start 6-25) |
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| Success. (Like we need more EBFP2) Colonies picked and left on shaker at 2110 | hEF1a ENTR vector transformed into competent cells and plated onto Kan+ plates. |
14-6-26
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| id | 6-26 |
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| Propagation MAV1212-EBFP2 (Start 6-24) | Propagation hEF1a (Start 6-25) |
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Culture miniprep-ed | Colonies picked into LB-Kan medium. |
14-6-27
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| id | 6-27 |
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| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
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Golden Gate miR sensing sites onto MAV1212-EBFP2, replacing LacZ. Thermo-cycler started 2220 |
14-6-30
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| id | 6-30 |
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| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
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Product transformed into competent cells. |
14-7-1
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| id | 7-1 |
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| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
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| No colonies for GH. Two colonies picked from EF and incubated starting at 2112. Additional transformations started for both. Transformation plated and incubated starting at 1812. |
14-7-2
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| id | 7-2 |
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| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
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Mini-preped EF colonies. |
| PCR NcoI-mRFP-NsiI | Digest NcoI-mRFP-NsiI | Ligation |
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| Primers arrived. PCR products PCR-ed some more. | mRFP-PCR product digested concurrently with NcoI and NsiI. Digestion begun at 1922. | mRFP-PCR-product digestion is ligated with digested MAV-1212. |
| MAV1212-RFP Propagation (7-2) |
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| MAV1212-mRFP ligation products transformed into cells. Plated and left in the incubator at 0043 of next day. |
14-7-3
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| id | 7-3 |
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| MAV1212-RFP Propagation (7-2) |
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| Plated some more cells. Left in incubator at 2015 |
14-7-7
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| id | 7-7 |
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| MAV1212-RFP Propagation (7-2) |
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Red (actually barely pinkish) colonies, hallelujah. |
14-7-8
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| id | 7-8 |
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Cultures Minipreped.
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| Colony | 1 | 2 | 3 | 4 |
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| DNA | 172.1 | 171.4 | 134.9 | 281.6 |
| µL Added | 5 | 5 | 5 | 3 |
| Water | 12 | 12 | 12 | 14 |
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14-7-9
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| id | 7-9 |
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Halleluiah for Sample 4.
Propagate like crazy.
14-7-10
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| id | 7-10 |
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Transformations
miRNA:
MAV1212-mRFP
BP Product pENTR-TAL14/UAS Promoter
pENTR-L7ae
Treatment:
Golden Gate Product YFP-BACE1
BCR:
- LR: cd79b-TCS-G4
- LR: Gmab-heavy
- LR: Gmab-light
- LR: cd79b-Stop
- pEXPR-hef1a-cd79a-stop
- pEXPR-hef1a-cd79a-TCS-G4
- pEXPR-TER-sic-TEV
- pEXPR-TER:sic-stop
14-7-11
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| id | 7-11 |
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