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14-7-1

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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

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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

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MAV1212-RFP Propagation (7-2)
Plated some more cells.
Left in incubator at 2015

 

 

 

14-7-7

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 MAV1212-RFP Propagation (7-2)

Red (actually barely pinkish) colonies, hallelujah.
Colonies picked for shaker at 2035

 

 

14-7-8

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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

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Virtual GelReal Gel

 

 

 

Halleluiah for Sample 4.

Propagate like crazy.

 

 

14-7-10

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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

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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

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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

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Transformed LR products

 




Digested MAV1212-Hef1a-L7ae with BsmBI in 3.1 Buffer