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Zulu Time All.
14-6-17
| 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
| 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
| 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
| 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
| 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
| 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
| 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.
Plates incubated at 30°C incubator from 2120. |
14-6-26
| Propagation MAV1212-EBFP2 (Start 6-24) | Propagation hEF1a (Start 6-25) |
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Culture miniprep-ed
Concentration: | Colonies picked into LB-Kan medium.
Placed into shaker at |
14-6-27
| 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
| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
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Product transformed into competent cells.
New plates made with Amp and X-gal.
Incubation started at 2020. |
14-7-1
| 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
| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
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Mini-preped EF colonies.
40/50 ng/µL. |
| 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
| MAV1212-RFP Propagation (7-2) |
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Plated some more cells.
Left in incubator at 2015 |
14-7-7
| MAV1212-RFP Propagation (7-2) |
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Red (actually barely pinkish) colonies, hallelujah.
Colonies picked for shaker at 2035 |
14-7-8
| MAV1212-RFP Propagation (7-2) |
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Cultures Minipreped. Digested for Gel.
| Colony | 1 | 2 | 3 | 4 |
|---|
| DNA | 172.1 | 171.4 | 134.9 | 281.6 | | µL Added | 5 | 5 | 5 | 3 | | Water | 12 | 12 | 12 | 14 |
|
| Virtual (Ideal) | Virtual (Problematic) | Gel |
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 |  |  |
14-7-9
| Virtual Gel | Real Gel |
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| 
Halleluiah for Sample 4. Propagate like crazy. |
14-7-10
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
Picked colonies for:
MAV1212-mRFP
pENTR-L1-TALER14-L2
pEXPR-pTAL14-mKate-pEST
MAV1212-Hef1a-eBFP2
EF low sensor
Mutant Hef1a-2xKturn-eGFP
WT Hef1a-2xKturn-eGFP
14-7-16
