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14-7-1
| Golden Gate Variable Expression Vector: Low Sensor (6-27) |
|---|
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) |
|---|
Mini-preped EF colonies.
40/50 ng/µL. |
| PCR NcoI-mRFP-NsiI | Digest NcoI-mRFP-NsiI | Ligation |
|---|
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) |
|---|
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) |
|---|
Plated some more cells.
Left in incubator at 2015 |
14-7-7
| MAV1212-RFP Propagation (7-2) |
|---|
Red (actually barely pinkish) colonies, hallelujah.
Colonies picked for shaker at 2035 |
14-7-8
| MAV1212-RFP Propagation (7-2) |
|---|
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 |
|---|
 |  |  |
14-7-9
| Virtual Gel | Real Gel |
|---|
| 
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 |
|---|
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
