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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 |
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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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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
14-7-15
LR Gateway Reactions |
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MAV1212 + pENTRE-L4-TRE-tight-L1 + pENTRE-L1-Kozak-L7Ae-L2 = MAV1212-TRE-L7Ae MAV1212 + pENTRE-L4-TRE-tight-L1 + pENTRE-L1-GAL4VP16-L2 = MAV1212-TRE-L7Ae MAV1212 + pENTRE-L4-Hef1a-L1 + pENTRE-L1-GAL4VP16-L2 = MAV1212-TRE-L7Ae |
14-7-16
Transformed LR products
Digested MAV1212-Hef1a-L7ae with BsmBI in 3.1 Buffer
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