Alice:LabNotes/2009-12-5

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Solexa sequencing library preparetion[edit]

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.
  • 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
CViB: 54ng/ul

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 2009-12-06 14hr 17min.jpg

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

Ligation[edit]

  • Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.
  • 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.
End-repaired & size selected DNA 36ul
40uM adaptor2 2ul
5X Quick Ligase Buffer 10ul
Quick Ligase 2ul
  • 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
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

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)
  • Mix the amplicons with two sets of primers
  • Purified with Minelute columns, elute with 32ul EB buffer

TBE Gel Size Selection[edit]

  • Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel
  • Load 1 lane with 25bp ladder (20ul H2O+20ul 6X loading dye+1ul 25bp ladder)/2
  • Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells
File:ZhangLab 2 2009-12-07 21hr 35min.jpg