Alice:LabNotes/2009-10-28
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Solexa single-end sequencing library construction[edit]
- using NEBNext DNA Sample prep master mix I
- The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp-150bp.
End-repair Reactions[edit]
Fragmented DNA 85 ul 10X End Repair Reaction Buffer 10 ul End Repair Enzyme Mix 5 ul Keep the tube at room temperature (~20°C) for 30 minutes. Purify with QiaQuick columns, elute with 39ul buffer EB. 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[edit]
Blunt-end DNA 37 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul Klenow Fragment (3’-5’ exo-) 3 ul H2O 5 ul Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 42 ul EB
Nanodrop: 9-Cvi: 18.3ng/ul 8-cvf: 28ng/ul 8-NA: 23.8ng/ul 8-CVi: 22.7ng/ul 9-NA: 34ng/ul 9-CVf: 12.6ng/ul
E-gel size selection[edit]
Load each end-polished DNA sample into two lanes (use 3 or more lanes if the starting DNA is more than 1ug) of the Invitrogen 2% SizeSelect gel, also load 0.25ug of the Invitrogen 25bp ladder into another lane. Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the 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. Concentrate the DNA with a SpeedVac to ~36ul if necessary (do not turn the heater on). File:ZhangLab 2 2009-10-28 15hr 26min.jpgFile:ZhangLab 2 2009-10-28 16hr 00min.jpg File:ZhangLab 2 2009-10-28 16hr 35min.jpg
adaptor ligation[edit]
Prepare adaptors (need to be done only for the first time): Anneal adaptors by mixing 20 ul of 100 uM of the corresponding _up and _lo oligonucleotides (each) with 10ul 10X Taq buffer (or Stoffel buffer) and 50ul H2O, heating to 94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec.
Blunt-end adaptors: 5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH Solexa_1_up 3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH Solexa_1_lo_nop TA adaptors: 5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH PE_t_adapter 3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos t_adaptor_rc_s
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 36 ul 40uM adaptor 2 ul 5X Quick ligase buffer 10 ul Quick Ligase 2 ul
Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB.
PCR[edit]
Ligation products 10 ul 10ul 10uM Solexa_PCR_up 2 ul 2ul 10uM AmpR6.3Sol 2 ul - 10uM AmpF6.3rSol - 2ul 2X iProof master mix (Bio-Rad) 50ul 50ul 50X SYBR Green I 0.4ul 0.4ul H2O 36ul 36ul PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. purify the products with Minelute columns and elute in 15ul of EB. Then mix the two sets of products together. PAGE size selection : 200-275bp Ethanol precipitation and elute in 20ul H2O