Noi/NOTES/2011-9-1

From ZhangLabWiki
Revision as of 14:12, 2 September 2011 by >Noi (→‎PCR set up)
Jump to navigation Jump to search

Randomly tagging primer test

  • Received 4 new primers for the project
    • AmpF7NU.Sol
    • AmpF7AU.Sol
    • Syb_FP5A
    • Syb_RP7

Tests:

  • Does KAPA work?
  • Does new F primer work for both Phusion and KAPA?
  • Can we reduce primer conc. From 0.2uM to 0.02uM?

PCR set up

  • Phusion, 0.2uM primer, 3 tubes of reactions : 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC
  • Note: NTC used old F primer (AmpF6.4.Sol)
Components 1 rxn 3.1x rxn mix P
Circular DNA 2.00 0.00
10uM AmpF6.4Sol or new F 1.00 0.00
10uM AmpR6.3Sol 1.00 3.10
2x Phusion 25.00 77.50
50x SYBG 0.50 1.55
H2O 20.50 63.55
Total volume (ul) 50 145.70
  • aliquot 47ul, add 1ul of 10 uM F primer and 2ul of circular DNA

  • KAPA, 0.2uM primer, 3 tubes of reactions: 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC
  • Note: NTC used old F primer (AmpF6.4.Sol)
Components 1 rxn 3.1x rxn mix K
Circular DNA 2.00 0.00
10uM AmpF6.4Sol or new F 1.00 0.00
10uM AmpR6.3Sol 1.00 3.10
2x KAPA 25.00 77.50
H2O 21.00 65.10
Total volume (ul) 50 145.70
  • aliquot 47ul, add 1ul of 10uM F primer and 2ul of circular DNA

  • Phusion, 0.02uM primer 1 tube of reaction: old F primer
Components ==1 rxn==
Circular DNA 2.00
1uM AmpF6.4Sol 1.00
1uM AmpR6.3Sol 1.00
2x Phusion 25.00
50x SYBG 0.50
H2O 20.50
Total volume (ul) 50.00


  • KAPA, 0.02uM primer 1 tube of reaction: old F primer
Components ==1 rxn==
Circular DNA 2.00
1uM AmpF6.4Sol 1.00
1uM AmpR6.3Sol 1.00
2x KAPA 25.00
H2O 21.00
Total volume (ul) 50.00


Reaction codes

PCR conditions Reaction code
Phusion-NTC NTC-P
Phusion-old F primer-0.2uM primers P-o
Phusion-new F primer-0.2uM primer P-n
KAPA-NTC NTC-K
KAPA-old F primer-0.2uM primer K-o
KAPA-new F primer-0.2uM primer K-n
Phusion-old F primer-0.02uM P-o1
KAPA-old F primer-0.02uM primer K-o1


Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x25