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Date
Product
Gene
Starting Plasmid
Forward Primer
Reverse Primer

Thermocycler Variables: #cycles /

annealing temp / extension time

Gel Picture
6/23Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR30 / 60C / 30sGel Picture 6/23
Q1-eYFP-Q2eYFPpEXPR TRE:eYFPiGEM 018 eYFP N-term primerFiGEM 019 eYFP N-term primerR
Q1-BACE2-Q2BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR
Q2-eYFP-QXeYFPpEXPR TRE:eYFPiGEM 003-2 YFP C-term primerFiGEM 004-2 YFP C-term primerR
6/24Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR35 / 56.2C / 30sGel Picture 6/24
Q1-eYFP-Q2eYFPpEXPR TRE:eYFPiGEM 018 eYFP N-term primerFiGEM 019 eYFP N-term primerR
Q1-BACE2-Q2BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR
Q2-eYFP-QXeYFPpEXPR TRE:eYFPiGEM 003-2 YFP C-term primerFiGEM 004-2 YFP C-term primerR
6/25Q1-BACE2-Q2BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR

35 / 50.2C / 30s

 

 

 

 

 

Gel Picture 6/25
Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR
Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR
Q1-eYFP-Q2eYFPpEXPR TRE:eYFPiGEM 018 eYFP N-term primerFiGEM 019 eYFP N-term primerR
Q2-eYFP-QXeYFPpEXPR TRE:eYFPiGEM 003-2 YFP C-term primerFiGEM 004-2 YFP C-term primerR
6/26


Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR

30 / 55C / 1m

 

 

 

 

 




Gel Picture 6/26


Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR
Q1-BACE2-Q2BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR
Q1-BACE2-Q2BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR
6/26


   		Q1-BACE1-Q2BACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR

 

 

 

 

 

 




   


   
Q2-BACE1-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR
Q1-eYFP-Q2eYFPpEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR
Q2-eYFP-QXeYFPpEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR

 

 

 

Ran PCRs according to protocol on NEB website: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531

Used 20 uL reaction.  For plasmids, diluted to concentration 1ng/uL in EB buffer and added 1uL of each dilution to a separate PCR mix.  Diluted primers from 100uM stocks to 10uM with TE buffer.

 

PCR recipe:

Quantity
Chemical
1 uLforward primer (10uM)
1 uLreverse primer (10uM)
1 uLtemplate DNA (1 ng/uL)
10 uLPhusion High Fidelity PCR master mix
7 uLdH2O
20 uLTOTAL

 

Settings for thermocycler (program: PHUSION):

STEP 
TEMP 
TIME 
Initial Denaturation98°C30 seconds
30 Cycles98°C
60°C
72°C		5 seconds
30 seconds
30 seconds
Final Extension72°C5 minutes
Hold4°C 
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