Versions Compared

Old Version 10

changes.mady.by.user Unknown User (jamesa@mit.edu)

Saved on

compared with

New Version Current

changes.mady.by.user Unknown User (jamesa@mit.edu)

Saved on

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.
Forward Primer6/246/24Used 0.6 uL DMSO to reduce risk of hairpins or primer dimers
Date
Product
Gene
Starting Plasmid
Reverse Primer

Thermocycler Variables: #cycles /

annealing temp / extension time

Gel Picture

Comments / Deviations

from Protocol
6/23Q2-BACE2-QXBACE2pEXPR BACE2iGEM 016 BACE2 C-term primerFiGEM 017 BACE2 C-term primerR30 / 60C / 30sGel Picture 6/23Diluted primers in water, not TE
Q1-eYFP-Q2pEXPR TRE:eYFP6/23
Q1-eYFPBACE2-Q2pEXPR BACE2
Q2-eYFP-QXpEXPR TRE:eYFP
6/24Q2-BACE2-QXpEXPR BACE235 / 56.2C / 30sGel Picture 6/24
Q1-eYFP-Q2pEXPR TRE:eYFPiGEM 018 eYFP N-term primerFiGEM 019 eYFP N-term primerR30 / 60C / 30sPicture 6/23 Diluted primers in water, not TE6/23
Q1-BACE2-Q2pEXPR BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR30 / 60C / 30sPicture 6/23 Diluted primers in water, not TE6/23Q2-eYFP-QX
Q2-eYFP-QXpEXPR TRE:eYFP
6/25Q1-BACE2-Q2pEXPR BACE2

35 / 50.2C / 30s

 

 

 

 

 

Gel Picture 6/25
Q2-BACE2-QXpEXPR BACE2
Q2-BACE2-QXpEXPR BACE2
Q1-eYFP-Q2eYFPpEXPR TRE:eYFPiGEM 003-2 YFP C-term primerFiGEM 004-2 YFP C-term primerR30 / 60C / 30sPicture 6/23 Diluted primers in water, not TE
Q2-eYFP-QXpEXPR TRE:eYFP
6/26



Q2-BACE2-QX

(plus betaine)

pEXPR BACE2

30 / 55C / 1m

 

 

 

 

 





Gel Picture 6/26



Q2-BACE2-QX

(plus DMSO)

pEXPR BACE2

Q1-BACE2-Q2

(plus betaine)

pEXPR BACE2iGEM 016

Q1-BACE2

C-term primerF		iGEM 017 BACE2 C-term primerR35 / 56.2C / 30s Used 0.6 uL DMSO to reduce risk of hairpins or primer dimers

-Q2

(plus DMSO)

pEXPR BACE2
6/27Q2-BACE1-QXpEXPR BACE1 Gel Picture 6/27
Q2-eYFP-QXpEXPR eYFP
6/30Q1-eYFP-Q2pEXPR eYFPpEXPR TRE:eYFPiGEM 018 eYFP N-term primerFiGEM 019 eYFP N-term primerR35 / 56.2C / 30s Used 0.6 uL DMSO to reduce risk of hairpins or primer dimers
6/24Q1-BACE2-Q2BACE2pEXPR BACE2iGEM 001-2 BACE2  N-term primerFiGEM 002-2 BACE2 N-term primerR35 / 56.2C / 30s Used 0.6 uL DMSO to reduce risk of hairpins or primer dimers
6/24Q2-eYFP-QXeYFPpEXPR TRE:eYFPiGEM 003-2 YFP C-term primerFiGEM 004-2 YFP C-term primerR35 / 56.2C / 30s 
 Gel Picture 6/30
Q1-BACE1-Q2pEXPR BACE1
7/9   Q2-eYFP-QX (for BACE2)pEXPR eYFP

35 / 58C / 1m

with both DMSO and Betaine

     		Gel Picture 7/9     
Q1-eYFP-Q2 (for BACE2)pEXPR eYFP
Q2-eYFP-QX (for BACE1)pEXPR eYFP
Q2-BACE2-QXpEXPR BACE2
Q1-BACE2-QXpEXPR BACE2
Q1-BACE1-QXpEXPR BACE1
7/10(miRNA generator stuff) 

35 / 58C / 1m

with both DMSO and Betaine

Gel Picture 7/10
(20 PCR products in all) 
7/11(miRNA generator stuff)  30 / 61C / 30s Gel Picture 7/11
(12 PCR products in all) 
7/16(miRNA generator stuff)  30 / 63C / 30s

Gel Picture 7/16

 (12 PCR products in all)   

 

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.

...