Page History

added:

1uL of TRE-t (1) @ 5fM

1uL of EYFP @ 5fM

1uL of pDEST 12 @ 10fM

0.5uL of clonase

mixed by pipetting, a few air bubbles in aliquot.

left over night at room temperature

Purpose: practice

added:

1uL of Hef1a (1) @ 5fM

1uL of EBFP @ 5fM

1uL of pDEST 12 @ 10fM

0.5uL of clonase

mixed by pipetting, one air bubble in aliquot.

left over night at room temperature

Purpose: practice

• after both LR reactions (for JA and AL) from 6/12 incubated at room temp for 24 hours, the protocol was followed exactly
• the plated transformed e. coli were left in the 30 C incubator at 1:30pm

• the plated LR transformants from 6/13 yielded no colonies (after incubating constantly since 6/13); the plates were discarded
• two new transformations were performed following the protocol; the vectors that were transformed were pre-made stock pEXPR Hef1a:BFP and pEXPR TRE-t:eYFP
• the transformants were plated on LB-Amp plates and incubated at 30C overnight 

• the transformants from 6/16 yielded ample colonies; two colonies were picked from each plate (one for a MiniPrep and one for a Midiprep), inoculated into separate LB-Amp solutions, and incubated overnight
• all the primers for the eYFP-BACE2 fusion arrived today
  • these primers unfortunately were designed too long and had undesirably high melting temperatures (approximately Tm = 80C for each primer)
  • nevertheless, PCR was performed using the YFP primers in the hopes that they might still work; either way, the primers were edited in Geneious and re-ordered so that we will have primers with a lower, more ideal melting temperature
  • the PCR products of YFP were analyzed on the nano-drop and displayed a concentration of 299.3 ng/uL (I'm not sure how useful this metric is, since it could just be representing the template DNA and not any desired PCR products)
  • PCR was not performed on BACE2 because the BACE2 gene first needs to be mini-prepped out of the bacteria it was delivered in; these bacteria have been plated to incubate overnight
  • Gel of YFP PCR results:
    

• note: the BACE1 stock cell supply is in the "iGEM 2014" box in the -80C freezer on the second floor (215) in the tube with the yellow cap with the side sticker that reads "HsCD00043526"
• note: the BACE2 stock cell supply is in the "iGEM 2014" box in the -80C freezer on the second floor (215) in the tube with the green cap with the side sticker that reads "...somethingsomething76140"

Constructing pEXPR TRE:BACE2-eYFP and pEXPR TRE:eYFP-BACE2

• ran PCR on BACE2 and on eYFP to produce Q1-BACE2-Q2 and Q2-eYFP-QX and Q1-eYFP-Q2 and Q2-BACE2-QX
• order of lanes: ladder, Q2-eYFP-QX, Q1-eYFP-Q2, Q1-BACE2-Q2, Q2-BACE2-QX, (team B-cell receptor-->) 00798, FD798TOS, TCSGo14VP16

Constructing pEXPR Hef1a:BACE1-eYFP and pEXPR Hef1a:eYFP-BACE1

• there were no colonies on the BACE1 plate, so I re-plated the BACE1 cells on a Kan plate

- Troubleshooted PCR - considered lowering annealing temp, only doing one pcr at a time

Transferred BACE1 to liquid culture

• added 5mL LB to round bottom tube
• added 5uL of Kan
• picked colony
• placed tube in shaking incubator @ 37 C

PCR on BACE2 and eYFP

performed for both constructs (N and C term) of BACE2 and eYFP

• 6.4 uL H2O
• 1uL forward primer
• 1uL reverse primer
• 1uL template DNA @1ng/uL
• 0.6uL DMSO
• 10uL 2X Phusion master mix

Annealing temperature was determined from the NEB calculator to be 58C and 59C for the two constructs. The lower temperature was chosen to do all at the same time. Since this mix includes 3% DMSO, the annealing temp was lowered by 0.6C per 1% DMSO to 56.2C

Gel

Hyperladder 1kb | C eYFP | C BACE2 | N eYFP | N BACE2 @120V

Performed miniprep as per protocol on BACE1 liquid culture

Ran PCR on eyfp and bace2, both n and c term w/ dmso @50.2C

Ran gel, 1% agarose, 120V

PCR BACE2

• 1 uL forward primer
• 1 uL reverse primer
• 1 uL template @ 1ng/uL
• 4 uL betaine @ 5M
• 3 uL H2O
• 10 uL Phusion MM
  

30 cycles, 60 second extension time, 55C anneal temp

1 = C BACE2 w/ Betaine

2 = N BACE2 w/ Betaine

3 = C BACE2 w/ DMSO

4 = N BACE2 w/ DMSO

BACE1 and YFP PCR labeling code:

• A= C-YFP
• B= N-BACE1
• C= N-YFP
• D= C-BACE1

Ran Gel

PCR b,c

pcr purification - followed written protocol, warmed EB, added 10uL NaAC, 100uL PB

CBACE1 - 32.4 ng/uL

CEYFP1 - 26 ng/uL

CEYFP1 - 23 ng/uL

NEYFP2 - 30.3ng/uL

gg:

1.65 yfp

1.54 bace1

.8 gg donr

transformation of bpenter yfp bace1 and ggdonr control

made kan

LC yfp-bace1

pcr n-bace1, 1 w/ dmso 1w/ betaine 1 w/ both 30 cycles @53C

• restriction-digested ten colonies' mini-prepped DNA from the plate of bacteria transformed with the eYFP-BACE1 GG product
• restriction enzyme was BsrGI
• note: only 8 uL of DNA was used in each digest, regardless of DNA concentration; this means that none of the restriction digests technically received enough DNA (the protocol calls for at least 500 ng, whereas most of the GG product DNA was around 20-50 ng/uL)

miniprepped yfp-bace1

goldengate bace1-yfp:

nbace1: .95 ul

cyfp1: 2.2

gg donr: .88

h20: 9.97

restrition digest yfp-bace1 w/ ggdonr digest as control

picked bace2 for LC, incase original miniprep was screwed up

• The eBFP2 digest with BsrGI was a control to test whether BsrGI was working properly. If BsrGI were working properly we would have expected 3 bands. Yet there is no DNA of any length visible in this lane. I'm not sure of the reason for this but in any case this lane is useless in telling us whether or not BsrGI works. On Monday we will run a gel of non-digested GG product (eYFP-BACE1) to compare the gel pattern of undigested GG product vs digested GG product.
• The digests with PstI were a second attempt to validate proper GG assembly of eYFP-BACE1 (because our first attempt at restriction digesting with BsrGI was a dud). Yet oddly, we again see that no digestion occurred.

LR Bace1 and bace2, creating 5 fm concentration using the excel sheet on the protocol page.

ran gel of undigested gg donr and products.

L

 Code for today's miniprep samples:

• X = BACE2-YFP
• Y = BACE1-YFP
• Z = YFP-BACE2
• also note: W = YFP-BACE1

"XC" was discarded, so there will be only 9 miniprep samples forBACE2-YFP

 Ran restriction Digest on LR products. Used 5uL of tre bace2 a,b,c and hef1a bace1 a. 3 uL of hef1a bace1 c and 10 uL of hef1a bace1 b.

Digested with apa1 for bace1 and basaa1 for bace2

HL 1kb | TB2 A control | TB2 A | TB2 B | TB2 C | HB1 A control | HB1 A | HB1 B | HB1 C| Supercoiled ladder

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