Noi/NOTES/2013-4-30

From ZhangLabWiki
Jump to navigation Jump to search
Sample list (4ul each)
3 1ng UDG/Endo IV
3 10ng UDG/Endo IV
2 10ng USER/PNK
  • Manuals: [1]
  • Some additional info for library prep (USER digestion and PCR): [2]
  • Note: the manual didn't provide the concentration adapter and primers. I got the number from neb technical support
  • Adapter and primer info
    • NEBNext Adaptor
      • Loop adapter containing ideoxyU, need to be cleaved with USER after ligation.
      • Conc. 15uM. Total volume 240ul. I aliquoted 25ul in 9 striptubes
      • Since we used very little for ligtion (dilute 10X and add 1.67ul to each reaction for 1-10ng DNA input to get final conc. of adaptor in total 50ul reaction 0.05uM), this amount can be used to ligate ~1400 reactions
    • NEBNext Universal PCR Primer
      • Equivalent to PCR_F primer
      • Conc. 25uM. Total volume 70ul. I dilute to 10uM in total volume 175ul
    • NEBNext Index 1–12 Primers
      • Conc. 25uM. Total volume 12ul. I dilute to 10uM in total volume 30ul and transferred to striptube for handling with multichannel pipette
      • The sequences are different from PCR_R.N2Index (the same index but different in 3'- sequences. So if we used NEB adaptor, supposed to use reverse primer (NEBNext Index) from NEB.
PCR_R.N2Ind1:   5'- CAAGCAGAAGACGGCATACGAGATCGTGATCTCGGCATTCCTGCTGAACCGCTCTT -3'
NEBNext Index1: 5'- CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T  -3'
-s- = phosphorothioate bond
* HP1 primer for read1 is the same since the NEBNext Universal PCR Primer is equivalent to PCR_F
* Need to design new read2 primer (N2RevSeq2) --> NEBNextRevSeq2: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3' (Tm = 74.4 C)
* Need to design new index sequence read primer --> NEBNextIndSeq: 5'- GAAGAGCACACGTCTGAACTCCAGTCAC -3' (TM = 69.2 C)
  • All sequences of adapter and primer from NEB kit will be updated on the Illunima GA/Oligo info in the main page

Adapter ligation[edit]

  • I used the same concentration of adaptor as I did on April25, 2013
  • Adapter ligation mix (Total 50ul) : Ligation reaction mix 48.33ul/reaction & 1.67ul of 1.5uM adapters (Dilute 15uM adapters 10X with H2O)

Adapter preparation[edit]

  • Mix 2ul of 15uM loop adapter with 18ul H2O (final conc. 1.5uM).

Ligation reaction mix[edit]

Ligation reaction mix 1x rxn 9x rxn mix
KAPA 5X Ligation buffer 10.00 90.00
KAPA DNA ligase 5.00 45.00
H2O 19.33 173.97
Total 34.33 308.97
  • Aliquot 34.33 ul of ligation reaction mix to each tube
  • Transfer 4ul of sample to reaction tubes
  • As I mentioned before, if the sample is already in a clear tube, just add ligation reaction mix directly to fragmented DNA and adjust volume in ligation reaction mix
    • I used 0.2 ml PCR Tube Strips, low-Profile tube (Biorad) to set up reaction
  • Rinse the tube with 10ul H2O and transfer all to reaction tube (total volume now is 48.33ul)
  • Add 1.67ul of 1.5uM loop adaptor
  • 20C for 15min (no shaking)
  • Add 1.5ul USER (come with the kit), 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, so might increase volume of USER to 2ul/ reaction or use 1.5ul with longer incubation (~30min) for the completion of USER digestion
  • 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[edit]

  • I reduced extension time to 45 sec, and increased annealing temperature to 65C
Components 1x rxn 9.5 rxn mix
adapter ligated DNA 20.00 0.00
PCR_F (NEB, 10uM) 1.00 9.50
NEBNext Index (10uM) 1.00 0.00
2X KAPA SYBR MM 25.00 237.50
H2O 3.00 28.50
Total volume 50.00 475.00
  • Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Samples Indx
1ng UDG/Endo IV -1 NEBNext Index1
1ng UDG/Endo IV -2 NEBNext Index2
1ng UDG/Endo IV -3 NEBNext Index3
10ng UDG/Endo IV -1 NEBNext Index4
10ng UDG/Endo IV -2 NEBNext Index5
10ng UDG/Endo IV -3 NEBNext Index6
10ng USER/PNK -1 NEBNext Index7
10ng USER/PNK -2 NEBNext Index8
NTC NEBNext Index1
Program (Eppendorf Realplex)
98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C
File:20130430 lowinput CGI.png

* 1ng UDG/Endo IV : 13 cycles
* 10ng UDG/Endo IV : 10 cycles
* 10ng USER/PNK : 12 cycles
  • 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 2013-04-30 19hr 43min CGI qPCR.jpg
  • From the gel image, using NEB loop adaptor can remove most of the background from adaptor. The library size was reduced as expected since Jeff increased dUTP and got more amplification with smaller number of cycle.