Alice:LabNotes/2010-1-18: Difference between revisions

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*using NEBNext DNA Sample prep master mix I
*using NEBNext DNA Sample prep master mix I
*The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp-130bp.
*The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp-130bp.
*received 2 sheared capture products from Harvard in late Ocboter
*received the sheared capture products from Harvard in late Ocboter


===End-repair Reactions===
===End-repair Reactions===
Line 14: Line 14:


  Nanodrop result:
  Nanodrop result:
  PGP7:
  PGP7: 16.3ng/ul


===A-Tailing reactions===
===A-Tailing reactions===
Line 27: Line 27:
  PGP7: 2.3ng/ul
  PGP7: 2.3ng/ul


===adaptor ligation===
===adaptor ligation (1-21-10)===
  Prepare adaptors (need to be done only for the first time):  
  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
  Anneal adaptors by mixing 20 ul of 100 uM of the corresponding  _up and _lo oligonucleotides (each) with 10ul 10X Taq buffer (or Stoffel
Line 55: Line 55:


  Incubate at room temperature for 15 minutes
  Incubate at room temperature for 15 minutes
  purify with Ampure kit and eluted with 40ulddH2O
  purify with Ampure kit and eluted with 40ul ddH2O
 
Nanodrop result:
PGP7:


===PCR===
===PCR===
Line 72: Line 69:
  purify the products with Minelute columns and elute in 15ul of EB. Then mix the two sets of products together.
  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
  PAGE size selection : 200-275bp
[[File:ZhangLab_2 2010-01-21 11hr 27min.jpg]]
  Ethanol precipitation and elute in 20ul H2O
  Ethanol precipitation and elute in 20ul H2O
[[File:ZhangLab_2 2010-01-23 18hr 01min.jpg]]

Latest revision as of 19:01, 28 January 2010

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