Alice:LabNotes/2010-1-18

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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-130bp.
  • received the sheared capture products from Harvard in late Ocboter

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 Agencourt Ampure kit and elute in 40ul ddH2O
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.
Nanodrop result:
PGP7: 16.3ng/ul

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 the products with Agencourt Ampure kit and elute in 40ul ddH2O
Nanodrop:
PGP7: 2.3ng/ul

adaptor ligation (1-21-10)[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. 
commonly used adaptors:
Blunt-end adaptors:
5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH	 	Solexa_1_up
3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH	        Solexa_1_lo_nop

TA adaptors (for the one adaptor protocol):
5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH		PE_t_adapter
3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos	                t_adaptor_rc_s

regular Y adaptor:
PE_t_adaptor(top)              ACACTCTTTCCCTACACGACGCTCTTCCGATC*T              3'-Phosphorothioate bond	
PE_b_adaptor(bottom)           \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG       5'-phosphorylation	
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. 
adaptor:target molar ratio is 1:10~20
End-repaired & size selected DNA           36 ul
20uM TA adaptor                             2 ul
5X Quick ligase buffer                     10 ul
Quick Ligase                                2 ul
Incubate at room temperature for 15 minutes
purify with Ampure kit and eluted with 40ul ddH2O

PCR[edit]

Ligation products              15 ul        15ul
10uM Solexa_PCR_up              2 ul         2ul
10uM AmpR6.3Sol                 2 ul          -
10uM AmpF6.3rSol                 -           2ul
2X iProof master mix (Bio-Rad) 50 ul        50ul
50X SYBR Green I               0.4ul       0.4ul
H2O                            21 ul        21ul
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
File:ZhangLab 2 2010-01-21 11hr 27min.jpg
Ethanol precipitation and elute in 20ul H2O
File:ZhangLab 2 2010-01-23 18hr 01min.jpg