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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-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

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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.

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

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Virtual Gel

Real 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:

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

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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

14-7-17

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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

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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
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14-7-19

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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
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14-7-21

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Why why why why why

Found out that we have no low sensors.

14-7-22

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MAV1212-TRE-L7ae

Incomprehensible bands, recommend re-LR.

14-7-23

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Child pages

Versions Compared

Old Version 10

New Version 11

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