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Zulu Time All.

 

14-6-17

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Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Begun 6-17)

Stock Concentrations:

EBFP2: 1118 ng/µL

MAV: unknown.

1856: Diluted DNA samples for both x100 before transformation

4 Plates, LO HI for both.

2144: Culture plates incubated overnight at 30°C for 16-18 hours.

 

 

14-6-18

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Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Begun 6-17)
1426: Checked cell plates. EBFP2 produced colonies. MAV1212 overgrown.
MAV1212 Streaking (Start 6-18)Propagating EBFP2: Pick Colonies (Start 6-17)
2044: Picked single colonies from each plate and streaked over xgal plates.
Let on the incubator overnight at 30°C for 16-18 hours.		2040: Picked one colony each from the HI and LO plates and immersed in LB Media.
Left on the shaker incubator overnight.

 

 

14-6-19

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MAV1212 Streaking (Start 6-18) OVER
1254: Disaster. No colonies.
Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Start 6-19)

Concentrations:

EBFP2: 12 ng/µL

MAV: 4 ng/µL

 

4 Plates, LO HI for both.

1715: Culture plates inseerted into 30°C incubator.

 

 

14-6-20

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Propagating MAV1212 and MAV1212-Hef1a-EBFP2 (Start 6-19) OVER

1105: Low transformation rate. Suspicions fall on faulty plates. Plating to be redone later with existing samples and streaking.

 

 

14-6-23

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Propagating MAV1212: Colony Picking (Start 6-17)Propagation MAV1212 (Start 6-23)

Attempted to isolate monoclonal colony from MAV1212 lawn.

Inserted into Amp+ LB medium.

Left on incubator shaker at 2114.

Re-plated transformation suspension on Amp+ plates.

Streaked colonies from lawn.

Left on 30°C incubator at 2120.

 

 

14-6-24

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Propagating MAV1212: Colony Picking (Start 6-17) OVERPropagation MAV1212 (Start 6-23) OVER

Medium Clear.

Growth failed.

Current MAV1212 vectors abandoned due to overactive ccdB gene killing off cells.

Awaiting arrival of new vectors with better survivability.

Propagation MAV1212-EBFP2 (Start 6-24)

Transformed onto Amp+ plates.

Left on 30°C incubator at 2135.

 

 

14-6-25

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Propagation MAV1212-EBFP2 (Start 6-24)Propagation hEF1a (Start 6-25)
Success. (Like we need more EBFP2)
Colonies picked and left on shaker at 2110

hEF1a ENTR vector transformed into competent cells and plated onto Kan+ plates.
Plates incubated at 30°C incubator from 2120.

 

 

14-6-26

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Propagation MAV1212-EBFP2 (Start 6-24)Propagation hEF1a (Start 6-25)

Culture miniprep-ed
Concentration:

Colonies picked into LB-Kan medium.
Placed into shaker at

 

 

14-6-27

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Golden Gate Variable Expression Vector: Low Sensor (6-27)

Golden Gate miR sensing sites onto MAV1212-EBFP2, replacing LacZ.

Thermo-cycler started 2220

 

 

14-6-30

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Golden Gate Variable Expression Vector: Low Sensor (6-27)

Product transformed into competent cells.
New plates made with Amp and X-gal.
Incubation started at 2020.

 

 

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