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 |
---|
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 |
---|
![](/confluence/download/attachments/98622515/RFP-Good.PNG?version=1&modificationDate=1404852502000&api=v2) | ![](/confluence/download/attachments/98622515/RFP-Bad.PNG?version=1&modificationDate=1404852541000&api=v2) | ![](/confluence/download/attachments/98622515/RFP-2.jpg?version=1&modificationDate=1404852136000&api=v2) |
14-7-9
Virtual Gel | Real Gel |
---|
![](/confluence/download/attachments/98622515/RFP-Good.PNG?version=1&modificationDate=1404852502000&api=v2) | ![](/confluence/download/attachments/98622515/MAV1212-RFP-7-9.png?version=2&modificationDate=1404952256000&api=v2)
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
![](/confluence/download/attachments/98622515/MAV1212-hef1a-L7ae.png?version=1&modificationDate=1405604971000&api=v2)
14-7-17
Miniprep and Midiprep | Transformations |
---|
MAV1212-Hef1a-Gal4VP16 MAV1212-TRE-Gal4VP16 MAV1212-TRE-L7ae | MAV1212-Hef1a-eBFP2-miR181 MAV1212-Hef1a-eBFP2-miR144 MAV1212-Hef1a-L7Ae-miR146 MAV1212-Hef1a-L7Ae-miR7 MAV1212-Hef1a-L7Ae-miR125 MAV1212-Hef1a-L7Ae-miR30 |
14-7-18
DNA | Con (ng/µL) | Enzyme | Buffer | Vol DNA | Mass DNA | Vol Enzyme | Vol Buffer | Vol Water | Vol Total |
Ladder | | | | | | | | | |
MAV1212-Hef1a-Gal4VP16 | 305.5 | BsaI | 3.1 | 2.45 | 750 | 1 | 2.00 | 14.55 | 20 |
MAV1212-Hef1a-Gal4VP16 control | 305.5 | | | 2.45 | 750 | | | 17.55 | 20 |
MAV1212-TRE-Gal4VP16 | 74.3 | BsaI | 3.1 | 10.09 | 750 | 1 | 2 | 6.91 | 20 |
MAV1212-TRE-Gal4VP16 control | 74.3 | | | 10.09 | 750 | | | 9.91 | 20 |
MAV1212-TRE-L7ae | 58.7 | BsaI | 3.1 | 12.78 | 750 | 1 | 2 | 4.22 | 20 |
MAV1212-TRE-L7ae control | 58.7 | | | 12.78 | 750 | | | 7.22 | 20 |
MAV1212-Hef1a-eBFP2-miR181 | 118.6 | BsaI | 3.1 | 6.32 | 750 | 1 | 2 | 10.68 | 20 |
MAV1212-Hef1a-eBFP2-miR181 control | 118.6 | | | 6.32 | 750 | | | 13.68 | 20 |
MAV1212-Hef1a-eBFP2-miR144 | 49.9 | BsaI | 3.1 | 15.03 | 750 | 1 | 2 | 1.970 | 20 |
MAV1212-Hef1a-eBFP2-miR144 control | 49.9 | | | 15.03 | 750 | | | 4.97 | 20 |
Ladder | | | | | | | | | |
![](/confluence/download/attachments/100213565/MAV1212-hef1a-Gal4VP16.png?version=1&modificationDate=1405950332000&api=v2)
![](/confluence/download/thumbnails/100213565/MAV1212-TRE-Gal4VP16.PNG?version=1&modificationDate=1405950596000&api=v2)
14-7-19
DNA | Con (ng/µL) | Enzyme | Buffer | Vol DNA | Mass DNA | Vol Enzyme | Vol Buffer | Vol Water | Vol Total |
Ladder | | | | | | | | | |
MAV1212-TRE-Gal4VP16 | 74.3 | BsaI | 3.1 | 6.73 | 500 | 1 | 2 | 10.27 | 20 |
MAV1212-TRE-Gal4VP16 control | 74.3 | | | 6.73 | 500 | | | 13.27 | 20 |
MAV1212-TRE-L7ae | 58.7 | BsaI | 3.1 | 8.52 | 500 | 1 | 2 | 8.48 | 20 |
MAV1212-TRE-L7ae control | 58.7 | | | 8.52 | 500 | | | 11.48 | 20 |
MAV1212-Hef1a-eBFP2-miR181 | 118.6 | BsaI | 3.1 | 4.22 | 500 | 1 | 2 | 12.78 | 20 |
MAV1212-Hef1a-eBFP2-miR181 control | 118.6 | | | 4.22 | 500 | | | 15.78 | 20 |
MAV1212-Hef1a-eBFP2-miR144 | 632.6 | BsaI | 3.1 | 1.581 | 1000 | 1 | 2 | 15.42 | 20 |
MAV1212-Hef1a-eBFP2-miR144 control | 632.6 | | | 1.581 | 1000 | | | 18.42 | 20 |
MAV1212-Hef1a-eBFP2 | 50.3 | BsaI | 3.1 | 9.94 | 500 | 1 | 2 | 7.06 | 20 |
MAV1212-Hef1a-eBFP2 control | 50.3 | | | 9.94 | 500 | | | 10.06 | 20 |
Ladder | | | | | | | | | |
![](/confluence/download/attachments/100213565/7-19.png?version=1&modificationDate=1405950453000&api=v2)
14-7-21
Why why why why why
Found out that we have no low sensors.
![](/confluence/download/attachments/100213565/7-21.png?version=1&modificationDate=1405988298000&api=v2)
![](/confluence/download/thumbnails/100213565/MAV1212-TRE-Gal4VP16.PNG?version=1&modificationDate=1405950596000&api=v2)
14-7-22
MAV1212-TRE-L7ae
![](/confluence/download/attachments/100213565/terribad.jpg?version=1&modificationDate=1406122386000&api=v2)
Incomprehensible bands, recommend re-LR.
14-7-23
![](/confluence/download/attachments/100213565/Screen%20Shot%202014-07-23%20at%202.50.43%20PM.png?version=1&modificationDate=1406141575000&api=v2)
![](/confluence/download/thumbnails/100213565/IMG_20140723_160637.jpg?version=1&modificationDate=1406148398000&api=v2)
weeeeee!
14-7-24
MAV1212-Hef1a-TAL14
EcoRI
![](/confluence/download/attachments/100213565/7-24.jpg?version=2&modificationDate=1406235402000&api=v2)
![](/confluence/download/attachments/100213565/Screen%20Shot%202014-07-23%20at%202.50.43%20PM.png?version=1&modificationDate=1406141575000&api=v2)
14-7-25
Golden Gate |
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Combined Low Sensor |
14-7-26
Midi Prep |
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MAV1212-Hef1a-GAL4VP16 MAV1212-TRE-GAL4VP16 Single Low Sensors |
Transformation |
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MAV1212-Hef1a-eBFP2-miR144-181c |
14-7-28
![](/confluence/download/attachments/100213565/MAV1212-TRE-L7ae.png?version=1&modificationDate=1406641431000&api=v2)