EricChu:RepliPHI MDA protocol (20ul reaction): Difference between revisions

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>Ericchu
(Created page with "==RephiPHI MDA Protocol (20ul reaction)== #Start with 1.0ul λ DNA (2.5ng/ul) #Add 1.5ul ALS, incubate at room temp 3mins #Add 1.5ul NS, put on ice immediately #Create master...")
 
>Ericchu
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==RephiPHI MDA Protocol (20ul reaction)==
==RephiPHI MDA Protocol (20ul reaction)==


#Start with 1.0ul λ DNA (2.5ng/ul)
#Start with 1.0ul λ DNA (2.5ng/ul) in PCR tube
#Add 1.5ul ALS, incubate at room temp 3mins
#Add 1.5ul ALS, incubate at room temp 3mins
#Add 1.5ul NS, put on ice immediately
#Add 1.5ul NS, put on ice immediately
Line 11: Line 11:
##2ul 8% Tween20  
##2ul 8% Tween20  
##4.2ul H20
##4.2ul H20
#Add master mix to PCR tube
#Add to thermocycler for 10 hours at 30⁰C and keep at 4 ⁰C
#Use PCR purification kit to collect DNA
#Measure DNA concentration with NanoDrop


ALS: 69.5ul H20 + 10ul DTT(1M) + 8ul KOH (5M) + 12.5ul 8% Tween20
ALS: 69.5ul H20 + 10ul DTT(1M) + 8ul KOH (5M) + 12.5ul 8% Tween20

Latest revision as of 23:38, 4 February 2013

RephiPHI MDA Protocol (20ul reaction)[edit]

  1. Start with 1.0ul λ DNA (2.5ng/ul) in PCR tube
  2. Add 1.5ul ALS, incubate at room temp 3mins
  3. Add 1.5ul NS, put on ice immediately
  4. Create master mix (w/ 1% Tween20)
    1. 2ul 10x buffer
    2. 5ul 200uM N6 primers
    3. 0.8ul 25mM dNTPs
    4. 2ul Phi29 polymerase
    5. 2ul 8% Tween20
    6. 4.2ul H20
  5. Add master mix to PCR tube
  6. Add to thermocycler for 10 hours at 30⁰C and keep at 4 ⁰C
  7. Use PCR purification kit to collect DNA
  8. Measure DNA concentration with NanoDrop

ALS: 69.5ul H20 + 10ul DTT(1M) + 8ul KOH (5M) + 12.5ul 8% Tween20

NS: 87.5ul NS stock + 12.5ul 8% Tween20

where NS stock: 4ml 1MHCl and 6ml 1MTrisHCl