Page History

Here's the order for the lanes of the four gels through which we ran our digested GG products:

1kb ladder | Supercoiled ladder | undig. GG donor | undig. GG product | BlpI-digested GG product | x-digested GG product | digested GG products A–J | 1kb ladder (optional)

Note the four gels were arranged in the order:

W Y

X Z

Six of the PCR screen products were identified as potentially being our desired product, and were transformed: XB8, XB4, YA9 ,YB3, YB4, ZB9.

Tasks for Saturday:

1. Stop the LR reaction and store in fridge
2. Miniprep miRNA1 and miRNA2
3. Pick colonies? (maybe not; pick Brian's at least)

PCR Screen Gels:

W: YFP-BACE1

X BACE2-YFP

YABACE1-YFP

YBBACE1-YFP

ZAYFP-BACE2

ZBYFP-BACE2

PCR Screen re-transformations:

miRNA Generator 4:

Tasks for Saturday:

• kill the LR of miRNA2b and transform
• miniprep the liquid culture of TRE:miRNA1

Tasks for Sunday:

• LR miRNA3 (either B,C,E, or G; they're all good) and miRNA4J each with TRE
• miniprep the liquid culture of TRE:miRNA1 (samples A through E)
• pick colonies from Saturday's transformation of TRE:miRNA2 (plate is in the 37C incubator on the second floor)

Digest of Hef1a:BACE1 and TRE:BACE2:

Re-digest of Hef1a:BACE1 and TRE:BACE2:

Digest of miRNA Generator 1 with ApaLI and XbaI

HL1 Kb | Undig. Prod| ApaLI | XbaI | ApaLI and XbaI A-E

Note: single digest with XbaI should result in a single band at 8 Kbp

Transfection notes:

• 1ug Hef1a:BACE1
• 1ug TRE:BACE2
• 2ug Hef1a: rtTA
• 3ug Hef1a:eBFP
• 3ug Dummy DNA
• 500uL diluted OptiMEM-lipofectamine

Neuronal research–primary neuron cultures and APPSwe HEK293:

• ease of culturing
• accuracy of models
• where do we get them
• pros/cons of use

miRG1 (BFP transfection marker) w/ red channel cranked

miRG1 + BACE1 (BFP transfection marker) w/ red channel cranked

miRG 2-4 expression digest

HL 1Kbp | undig. prod. | ScaI | XbaI 2-4 | miRG 2 A-E | miRG 3 A-E | miRG 4 A-E | HL 1Kbp

Hef1a:mKate + Hef1a:eBFP

Tre:miRG1 + Hef1a:eBFP + 1000nM Dox

 Sent the B samples of miRNAg2–4 for sequencing

 FRET Data

• Well A4 was not transfected with anything because Hef1a:miRNAg1 was not yet midiprepped.
• A3 also was not transfected (woops)

HL 1Kbp | undig. prod. | Hef1a:miRG1 A-E