AlanFung:LabNotes/Sequencing/2009-11-16

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Revision as of 19:15, 16 November 2009 by >Alan6017518 (→‎CVF-N)
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CVF-N

  • Reamplify library and ship it out for shearing
x4 X1.1
Library 12 0.25
2X Phusion 50 1.04 4.17 4.58
AmpF6.3NH2 (10uM) 2 0.04 0.17 0.18
AmpR6.3NH2 (10uM) 2 0.04 0.17 0.18
10X SYBR Green I 2 0.04 0.17 0.18
dH2O 32 0.67 2.67 2.93
  • 98C 1min->6X(98C 10 sec, 58C 20 sec, 72C 20sec)->72C 5 min

Construction of Solexa sequencing library from padlock captured PCR products using NEBNext DNA Sample Prep Master Mix Set 1 Kit (E6040S/L)

  • Info Read the FAQ!!!
  • Protocol
  • The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II.
  • For 60bp run, we can use the library size of >180bp
  • For 80bp runs, the lower limit for the sequencing libraries should be 200bp.
  • If the library size is too small, some of the 80bp reads will reach to the other end of the adaptor sequences. we don't want to waste the sequencing $$ on the regions we don't want
  • The total size of the adaptors is 105bp, and there is another ~25bp of the capturing arm
  • The upper limit should be ~50bp above the lower limit

Overview

1-7 PGP1,8 PGP1 & 9 PGP1

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


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

  • The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
  • 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

Size selection using Invitrogen 2% SizeSelect gel

  • 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 2009-11-05 14hr 35min.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.

  • 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

Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC

AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA

  • prepare 2 master mix tube
F R
10uM Solexa_PCR_up 18 18
10uM AmpR6.3Sol - 18
10uM AmpF6.3Sol 18 -
2X Phusion 450 450
50X SYBR Green I 3.6 3.6
H2O 324 324


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
CVI-F
CVI-R
CVF-F
CVF-R
1-7 PGP1-iPS-F
1-7 PGP1-iPS-R
9 PGP1-iPS-F
9 PGP1-iPS-R


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

  • TBE Gel verification (10well)
  • 15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
  • 15ul h2o+3ul 6X loading dye+2ul sampleFile:ZhangLab 2 2009-11-06 13hr 42min.jpg
  • Mix the amplicons with two sets of primers
  • Purified with Minelute columns, elute with 32ul EB buffer

TBE Gel Size Selection

  • 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