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 Transformation 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 Transformation 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