Noi/NOTES/2013-5-22

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Revision as of 22:08, 22 May 2013 by >Noi
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Ligation of CoRE fragmented DNA from Eric

  • Got 16 samples from Eric for ligation using NEBNext adaptor. (Samples were already in 0.2 ml PCR Tube Strips, low-Profile tube (Biorad))
  • Sample information should be noted by Eric.
Fragmented DNA 6.00
1.5uM loop adaptor 1.67
Ligation reaction mix 42.33
Total 50.00

Ligation reaction mix

Ligation reaction mix 1x rxn 16x rxn mix
2X KAPA Rapid Ligation Buffer 25.00 400.00
KAPA Rapid T4 DNA Ligase 2.00 32.00
H2O 15.33 245.28
Total 42.33 677.28
  • Aliquot 42.33 ul of ligation reaction mix to each tube containing 6ul of fragmented DNA
  • Add 1.67ul of 1.5uM NebNext loob adaptor. Mix well by pipetting 15-20 times and quick spin.
  • 25C for 10min (no shaking).
  • Add 2ul USER. Mix well by pipetting 15-20 times and quick spin. 37C for 15min
    • For the library prep using NEBNext® DNA Library and this loop adaptor, they used 3ul USER in total volume 50ul or 83.5ul, 37 C for 15 min. I used a little less.
  • Bead purification:
    • 50ul AmPure beads mix by pipetting 10 times
    • Wait 10min (no shaking)
    • Sit on magnetic stand 5min
    • Wash twice with freshly prepared 80% EtOH (after adding 80% EtOH, I wait for 30sec before pipette up and down few times and discard spnt)
      • I wash with 160ul of 80% EtOH since I used low profile tube.
    • Dry the beads for 3 min
  • Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR)

Amplification

Components 1x rxn 16.5 rxn mix
adapter ligated DNA 20.00 0.00
PCR_F (NEB, 10uM) 1.00 16.50
PCR_R.N2IndX (NEB, 10uM) 1.00 0.00
2X KAPA SYBR MM 25.00 412.50
H2O 3.00 49.50
Total volume 50.00 825.00
Samples Indx Samples Indx
#1 NEBNext Index1 #9 NEBNext Index1
#2 NEBNext Index2 #10 NEBNext Index2
#3 NEBNext Index3 #11 NEBNext Index3
#4 NEBNext Index4 #12 NEBNext Index4
#5 NEBNext Index5 #13 NEBNext Index5
#6 NEBNext Index6 #14 NEBNext Index6
#7 NEBNext Index7 #15 NEBNext Index7
#8 NEBNext Index8 #16 NEBNext Index8
  • Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Program (Eppendorf Realplex)
98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C
I stopped the reaction at 12 cycles
File:20130522 qPCR CoRE.png
  • Eric should provide the sample info.
  • PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume)
File:ZhangLab 2 2013-05-22 13hr 33min gel1 1-8.jpg  File:ZhangLab 2 2013-05-22 13hr 35min gel2 9-18.jpg