AlanFung:LabNotes/Sequencing/2009-11-25

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Overview[edit]

CVF-N(A-D Shearing), CVF-N (A,B USER), CVF-N (A-D USER)[edit]

  • Fragmentation and end-polishing
  • Size Selection
  • Ligation
  • PCR of sequencing Library
  • QPCR quantification


Fragmentation and end-polishing (Make blunt ends with 5'P)[edit]

  • End-Repair Reactions
Fragmented DNA 85ul
10X End Repair Bufer 10ul
End Repair Enzyme Mix 5ul
  • Incubate tubes at RT for 30minutes.
  • Perform a Qiaquick purification and elute with 39ul EB buffer.
  • NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
  • A-Tailing Reactions
Blunet-end DNA 37ul
10X dA-Tailing Reaction Buffer 5ul
Klenow Fragment (3'-5' exo-) 3ul
H2O 5ul

Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 40 ul EB.

  • Measure concentration with nanodrop
CVF-N USER A-B 3.8ng/ul
CVF-N USER A-D 3.3ng/ul
CVF-N Shearing 11.2ng/ul
  • Speed vac. the first 2 to ~20ul and run in 1 well in the size select gel

Size selection using Invitrogen 2% SizeSelect gel[edit]

  • Fill any unused well with 30ul EB Buffer
  • Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube
  • Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
  • Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well
  • Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
  • Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
  • Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.

File:ZhangLab 2 2009-11-25 17hr 16min.jpg

  • Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).

Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.[edit]

  • Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
ul
End-repaired & size selected DNA 36
40uM adaptor2 2
5X Quick Ligase Buffer 10
Quick Ligase 2
  • Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB.

PCR of sequencing library[edit]

Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC

AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA

  • prepare 2 master mix tube
F R
10uM Solexa_PCR_up 6.6 6.6
10uM AmpR6.3Sol 0 6.6
10uM AmpF6.3Sol 6.6 0
2X Phusion 165 165
50X SYBR Green I 1.32 1.32
H2O 118.8 118.8
Ligation products 10 10
10uM solexa PCR up 2 2
10uM AmpR6.3Sol 2 -
10uM AmpF6.3Sol - 2
2X Phusion 50 50
50X SYBR Green I 0.4 0.4
H2O 36 36
  • 90ul mix per well
  • Setup
CVF-N-F (Shearing)
CVF-N-R (Shearing)
CVF-N-F (A-B)
CVF-N-R (A-B)
CVF-N-F (A-D)
CVF-N-R (A-D)


PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.

  • TBE Gel verification (10well)
  • 10ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
  • 10ul h2o+3ul 6X loading dye+2ul sample

File:ZhangLab 2 2009-11-25 19hr 56min.jpg

  • Mix the amplicons with two sets of primers
  • Purified with Minelute columns, elute with 32ul EB buffer
CVF-N (Shearing) 18.1ng/ul *30ul=543ng
CVF-N (A-B) 27ng/ul *30ul=810ng
CVF-N (A-D) 38.8ng/ul *30ul=1164ng

TBE Gel Size Selection[edit]

  • Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel
  • Load 2 lanes with 25bp ladder Mix in a .2ml tube(30ul H2O+9ul 6X loading dye+1ul 25bp ladder)/2
  • Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells

File:ZhangLab 2 2009-11-25 22hr 15min.jpg File:ZhangLab 2 2009-11-25 22hr 16min.jpg File:ZhangLab 2 2009-11-25 22hr 18min.jpg