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-NsiIDigest NcoI-mRFP-NsiILigation
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.

Colony1234
DNA172.1171.4134.9281.6
µL Added5553
Water12121214
Virtual (Ideal)Virtual (Problematic)Gel

 

 

14-7-9

Virtual GelReal 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:

  1. LR: cd79b-TCS-G4
  2. LR: Gmab-heavy
  3. LR: Gmab-light
  4. LR: cd79b-Stop
  5. pEXPR-hef1a-cd79a-stop
  6. pEXPR-hef1a-cd79a-TCS-G4
  7. pEXPR-TER-sic-TEV
  8. 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

 

14-7-17

 

 

Miniprep and MidiprepTransformations

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

DNACon (ng/µL)EnzymeBufferVol DNAMass DNAVol EnzymeVol BufferVol WaterVol Total
Ladder         
MAV1212-Hef1a-Gal4VP16305.5BsaI3.12.4575012.0014.5520
MAV1212-Hef1a-Gal4VP16 control305.5  2.45750  17.5520
MAV1212-TRE-Gal4VP1674.3BsaI3.110.09750126.9120
MAV1212-TRE-Gal4VP16 control74.3  10.09750  9.9120
MAV1212-TRE-L7ae 58.7BsaI3.112.78750124.2220
MAV1212-TRE-L7ae control58.7  12.78750  7.2220
MAV1212-Hef1a-eBFP2-miR181118.6BsaI3.16.327501210.6820
MAV1212-Hef1a-eBFP2-miR181 control118.6  6.32750  13.6820
MAV1212-Hef1a-eBFP2-miR14449.9BsaI3.115.03750121.97020
MAV1212-Hef1a-eBFP2-miR144 control49.9  15.03750  4.9720
Ladder         

 

 

 

14-7-19

DNACon (ng/µL)EnzymeBufferVol DNAMass DNAVol EnzymeVol BufferVol WaterVol Total
Ladder         
MAV1212-TRE-Gal4VP1674.3BsaI3.16.735001210.2720
MAV1212-TRE-Gal4VP16 control74.3  6.73500  13.2720
MAV1212-TRE-L7ae 58.7BsaI3.18.52500128.4820
MAV1212-TRE-L7ae control58.7  8.52500  11.4820
MAV1212-Hef1a-eBFP2-miR181118.6BsaI3.14.225001212.7820
MAV1212-Hef1a-eBFP2-miR181 control118.6  4.22500  15.7820
MAV1212-Hef1a-eBFP2-miR144632.6BsaI3.11.58110001215.4220
MAV1212-Hef1a-eBFP2-miR144 control632.6  1.5811000  18.4220
MAV1212-Hef1a-eBFP250.3BsaI3.19.94500127.0620
MAV1212-Hef1a-eBFP2 control50.3  9.94500  10.0620
Ladder         

 

 

 

 

 

14-7-21

Why why why why why

Found out that we have no low sensors.

 

 

 

14-7-22

 

MAV1212-TRE-L7ae

Incomprehensible bands, recommend re-LR.

 

 

14-7-23

weeeeee!

 

 

 

14-7-24

 

MAV1212-Hef1a-TAL14

EcoRI

 

 

14-7-25

Golden Gate
Combined Low Sensor

 

 

 

14-7-26

Midi Prep

MAV1212-Hef1a-GAL4VP16
MAV1212-TRE-GAL4VP16
Single Low Sensors

Transformation
MAV1212-Hef1a-eBFP2-miR144-181c

 

 

14-7-28